Regulation by nutritional status of lipids and apolipoproteins A-I, A4, and A-IV in inbred mice

Transcription

1 Regultion by nutritionl sttus of lipids nd polipoproteins A-I, A4, nd A-IV in inbred mice R. C. LeBoeuf,' M. Cldwell, nd E. Kirk Deprtment of Medicine, University of Wshington, Settle, WA Abrtrct This study illustrtes tht genetic strin nd feeding sttus cn mrkedly influence tissue lipid concentrtions nd mrna levels of polipoprotein genes. C57BLJ6 nd BALBk mice were mintined for 2 weeks on four test diets differing in mount of cholesterol nd type of ft, nd fsted for 4 h or 16 h prior to collection of tissues. For both strins, the primry effect of fsting from 4 h to 16 h ws to prdoxiclly elevte triglyceride levels in plsm nd liver, nd to elevte heptic poa-iv mrna levels. 'Ifiglycefide secretion rtes, estimted fter the injection of Triton WR-1339, suggested tht elevtions in plsm triglyceride levels were due to reduced clernce of very low density lipoproteins. Although plsm glucose levels decresed with fsting time for both strins, insulin levels decresed for BALB/c but not C57BL/6 mice regrdless of diet. This suggests tht fctors thought to be medited by insulin, (e.g., plsm free ftty cid concentrtions; heptic pd-iv mrna levels) my be influenced by locl chnges in insulin sensitivity, which re controlled geneticlly nd re not reflected by plsm insulin levels. I In summry, nutritionl sttus influences constelltion of fctors involved in lipid trnsport tht lso show strong genetic components nd my influence subsequent nlyses of gene expression in the mouse system.- LeBocuf, B. C., M. Cldd, nd E. Kirk. Regultion by nutritionl sttus of lipids nd polipoproteins A-I, A-11, nd A- IV in inbred mice. J. Lipid RCS : Supplemcntuy key wordr mouse nutrition polipoprotein regultion genetics Inbred mice re proving to be vluble models for identifying genetic fctqrs in mmmlin plsm lipid trnsport, For instnce, hundreds of inbred strins re commercilly vilble nd mny hve been shown to exhibit substntil differences in plsm lipoprotein quntity, size, nd chemicl composition (1-3), polipoprotein regultion (4), nd ctivity of key enzymes in mmmlin lipid trnsport (5, 6). In ddition, the use of recombinnt inbred strin nd bckcross nlyses hve demonstrted the importnce of dietry-genetic interctions in the control of lipid trnsport (7) nd ssocited diseses (8, 9). The structurl genes for nerly ll of the mjor polipoproteins nd mny enzymes determining plsm lipoprotein structure nd concentrtion hve been mpped to mouse chromosomes (reviewed in ref. 9). Fi- nlly, geneticlly engineered mice with trnsgenes controlling the overexpression nd disruption of lipid trnsport genes hve been developed (reviewed in ref. 9). How nutritionl sttus influences the expression of lipoprotein trnsport genes in norml nd geneticlly modified strins is not entirely cler. In prticulr, the physiologicl consequences of food restriction on the expression of lipid trnsport proteins hs not been well chrcterized nd is the subject of this report. A common prctice in metbolic nd moleculr genetic studies is to collect tissues from mice fter n overnight (12-16 h) fsting period to void vritions in lipid prmeters due to postprndil fluctutions. However, food deprivtion in mmmls lters constelltion of physiologicl prmeters s tissues convert from utilizing glucose to fts nd eventully mino cids s fuel (1). Fuel conversion occurs rpidly (3-6 h) in smll mmmls such s mice nd rts s evidenced by mrked decreses in tissue glycogen levels (1, 11). Other chnges ssocited with semi-strvtion conditions include decreses in plsm insulin nd glucose levels nd increses in plsm FFA nd triglyceride concentrtions. Severl reports show tht modultion of heptic lipogenesis by food restriction lters the trnscriptionl ndor synthetic regultion of polipoprotein genes. In heptocytes tken from rts fsted for 3 dys (12), decresed rte of triglyceride secretion ws ccompnied by 5% decrese in pob-48 synthesis, lthough synthesis of pob-1 remined unchnged. In contrst, poe synthesis ws elevted 2- to 4-fold. The effect of fsting rts for 48 h on the mrna levels of severl plsm proteins showed mrked decreses in mrna for poa-11, more moderte decreses in mrna levels of poe, A-IV, lbumin, trnsferrin, nd trnsthyretin, nd 33% in- Abbrevitions: po, polipoprotein; FFA, free ftty cid; GAPDH, glycerldehyde-3-phosphte dehydrogense; HDL, high density lipoprotein; LDL, low density lipoprotein, "2, totl cholesterol; Triton, Triton WR-1339; VLDL, very low density lipoprotein. 'To whom correspondence should be ddressed. Journl of Lipid Reserch Volume 35,

2 crese in poa-i mrna (13). Poullin et l. (14) mesured plsm polipoprotein levels of rts fsted for 3 dys. While chnges in heptic pob mrna levels were dependent upon diet, poa-i nd A-IV mrnas showed mrked decreses nd poe mrna showed, mrked increse compred to fed nimls. Bum, Teng, nd Dvidson (15) showed tht refeeding rts fsted for 48 h with high crbohydrte diet resulted in 3-fold increse in heptic triglyceride content nd shift in pob editing from production of both B-1 nd B-48 to produce primrily pob-48. The refed rts lso exhibited moderte increses in poa-i, A-IV, nd E mrna levels. Thus, interprettion of gene regultion must include considertion of nutritionl sttus. Our lbortory is undertking systemtic study in mice to identify nd chrcterize genes susceptible to mnipultion by dietry cholesterol nd ftty cids. C57BL/6 nd BALB/c mice re used to model dietry responses in mice known to differ in therosclerosis susceptibility (8) nd other lipoprotein prmeters (1, 4). During the course of these studies, we observed mrked devitions in plsm lipid nd heptic polipoprotein mrna levels within ech individul mouse strin. In preliminry studies, differences in fsting times ppered to ccount for much of the intrstrin vrition. Thus in this report, we exmine severl prmeters of lipid trnsport in the plsm nd liver s function of two fsting times commonly used in the literture for mouse studies. We hve found tht fsting time drmticlly lters the plsm nd heptic concentrtions of triglyceride nd the mrna levels of polipoprotein A-IV in the liver. Animls MATERIALS AND METHODS Femle BALB/cByJ (BALBk) nd C57BL/6J (C57BLI6) mice from 6 to 8 weeks of ge were obtined from the Jckson Lbortory, Br Hrbor, ME. Mice were fed pelleted rodent chow diet (Wyne Rodent BLOX 864, Tekld, Mdison, WI) for 2 weeks prior to initition of diet studies. Mice were mintined in temperture-controlled (25OC) fcility with strict 12-h light/drk cycle. Mice were given free ccess to food nd wter. Food ws removed from the mice 4 h or 16 h prior to the collection of blood from the retroorbitl sinus into tubes contining nticogulnt nd ntimicrobil gents (1 mm E m, 5 kg/d gentmicin sulfte,.5% sodium zide). Plsm ws stored for l week or less t 4OC prior to nlysis. Mice were killed by cervicl disloction nd tissues obtined for lipid nlyses were plced directly into liquid nitrogen nd stored t -7OOC. This project ws pproved by the Animl Cre nd Use Committee of the University of Wshington (Protocol #214-1). Diets nd feeding Four diets were used in this study. Pelleted rodent chow nd n "therogenic" diet contining rodent chow known to elicit ftty strek lesions in strin C57BU6 mice (8) were obtined from Tekld Test Diets (Mdison, WI). The rodent chow diet contined pproximtely 4% ft, 24% protein, nd 4.5% crude fiber. The therogenic diet, described in detil by Pigen et l. (16), provided 3% kcl from ft (coco butter) nd contined 1.25% cholesterol nd.5% sodium cholte. Two high ft diets contining sfflower oil nd.5% sodium cholte with (sffhgh) nd without (sff/low) dded cholesterol (.5%) were prepred in our diet kitchen s described (17). The two sfflower oil diets were cloriclly blnced nd differed only in cholesterol content. These diets provided pproximtely 24% kcl from protein, 43% kcl from crbohydrte, nd 33% kcl from ft. A detiled description of dietry contents hs been given (17). Diets were stored in ir-tight continers t -2OOC nd fresh diet ws provided dily. Animl cceptnce of the semisynthetic diets ws improved by grdul introduction of diet over 4-dy period: 2 dys of 5:5 chow to semisynthetic diet, 2 dys of 2595 chow to semi-synthetic diet, followed by 2 weeks of 1% semi-synthetic diet (17). Mice were fed test diets during three seprte study periods owing to the vilbility of mice. In the first, mice were fed the therogenic diet for 2 weeks nd plsm nd tissues were collected s described bove. HDL cholesterol vlues were not obtined for these therogenic diet-fed mice due to insufficient volumes of plsm. The second feeding period consisted of mice fed rodent chow nd two sfflower oil-contining diets which were fed concurrently. HDL were seprted from other lipoproteins prior to the freezing of plsm smples for these mice. Plsm nd tissue nlyses s described below were determined for nimls of both sets of feeding studies simultneously. The third feeding study consisted of mice mintined on rodent chow for the Triton WR-1339 experiments s described below. Tissue lipid, glucose, nd insulin determintions Lipids were extrcted from mouse liver using the method of Folch, Lees, nd Slone Stnley (18), then modified to contin Triton X-1 s described by Crr, Andresen, nd Rude1 (19). The heptic nd plsm totl nd HDL cholesterol concentrtions were determined using colorimetric kit (Dignostic Kit, NO , Boehringer Mnnheim, Indinpolis, IN) with cholesterol stndrds (Preciset #125512, Boehringer Mnnheim). HDL cholesterol vlues were mesured fter the selective precipittion of VLDULDL by phosphotungstte (2). Triglyceride concentrtions in liver nd plsm were determined fter removl of free glycerol (Dignostic Kit, No. 4532, Boehringer Mnnheim). Heptic phos- 122 Joud of Lipid B ekd Volume 35, 1994

3 pholipids were determined ccording to Turner nd Rouser (21). Plsm free ftty cid quntities were determined using colorimetric kit (No , Wko Chemicls USA, Inc., Dlls, TX). Plsm glucose concentrtions were determined on Beckmn Glucose Anlyzer I1 utilizing glucose oxidse system. Plsm insulin quntifiction ws by rdioimmunossy (22) using n ntibody to guine pig insulin nd humn insulin stndrds. Apolipoprotein quntittion Plsm polipoprotein concentrtions were determined by slot-blotting. All ntibodies were determined to be monospecific by Western blot nlysis of mouse plsm. Rbbit ntiserum to poa-i ws prepred from isolted mouse poa-i. ApoA-I ws first seprted from other HDL proteins by Sephdex column chromtogrphy s described (23) nd further isolted from Coomssiestined bnd from 15% SDS-polycrylmide gels. Antiserum to poa-i1 ws prepred s described (23) nd results using this ntiserum lso described (2). ApoA-IV ws detected using ntiserum developed in rbbit to n poa- IV fusion protein (24) which ws generously provided by Deborh Purcell-Huynh nd Aldons J. Lusis (University of Cliforni, Los Angeles, CA). Plsm liquots (25 pl for poa-11, 5 pl for poa-iv, nd 5 pl for poa-i of plsm diluted 1:5) were pplied to nitrocellulose filters (.45 pm pore size, Schleicher nd Schuell, Keene, NH) using Minifold I1 slot-blotter (Schleicher nd Schuell) ccording to mnufcturers instructions. Filters were then removed from the blotter, ir-dried for 3 min, rinsed briefly in wter, nd soked for 15 min in 15% hydrogen peroxide. After brief rinse in distilled wter, filters were bked under vcuum for 1 h. Apolipoproteins were detected fter incubtion of filters with monospecific ntiser followed by incubtion with iodinted Protein A (2). Antiser dilutions of 1:lOOO were used. Signl intensities nd poa-iv were liner for volumes TABLE 1. rnging from 2.5 to 1 pl of plsm diluted by 1:5 with wter. For poa-i, linerity ws chieved in the rnge of 2.5 to 25 pl of plsm diluted by 1:5. This method routinely detected 5 ng of polipoprotein. Triton WR-1339 experiments Plsm triglyceride ccumultion with time ws ssessed following injection of Triton WR-1339 (designted in text s Triton; Tyloxpol, Sigm Chemicl Co., St. Louis, MO) vi the til vein of mice. Triton ws dissolved in.9% NCl (15% Triton w/v) nd injected t dose of pproximtely 3 mg/kg body weight between 1-11:3 AM fter fsting mice for either 4 h or 16 h. Blood smples for mesurement of triglyceride nd cholesterol were obtined t 3, 6, nd 12 min fter injection of Triton. Blood smples were lso tken t ech time point from mice injected with norml sline (shm injected). An liquot of plsm ws used to determine the concentrtion of Triton following precipittion of plsm proteins by 1:lO isopropnol nd spectroscopic reding t 278 nm s described (25). Triglyceride ccumultion rtes (Tble 1) were determined similrly to Ksim et l. (26) s: rte (mg/min) = 1/3 [(=~o-tgo)/~o + (TG6o-TGo)/6O + (TG12-TG)/12] x plsm volume, where X6, nd TG12 re triglyceride concentrtions t 3, 6, nd 12 min, respectively, nd TGo is triglyceride concentrtion for shm-injected mice (n = 3-6) used t ech time point. The plsm volume used ws estblished for mice t 5.77% of body weight (27). Messenger RNA quntittion Mouse cdna probes for poa-i nd poa-i1 were s described (2). A prtil mouse poa-iv cdna probe (7) ws generously provided by Deborh A. Purcell-Huynh nd Aldons J. Lusis (University of Cliforni, Los Angeles, CA). RNA ws isolted from mouse livers nd quntitted by Northern blotting s previously described Sttisticl prmeters describing effects of fsting time, diet, nd strin on plsm nd liver mesurements Plsm Liver Apolipoprotein ANOVA Fctors TC HDL-C TG Gluc Ins FFA A-I A-I1 A-IV A-I A-I1 A-IV C TG PL Time (T) < <.1 <.1 <.1 <.1.45 <.1 < < < Diet (D) <.1 CO.1 CO.1 CO <.1 <.1 < <.1 <.1 < Strin (S).115 <.1 < <.1 <.1.83 < <.1 <.1 <.1.8 T x D < T x S D x S <.1 CO.1 <.1 < <.1.6 <.1 < <.1 <.1.72 <.1 T x D x S.12.3 < < Tble bbrevitions re time (T), diet (D), strin (S), totl cholesterol (TC), HDL cholesterol (HDL-C), triglyceride (TG), glucose (Gluc), insulin (Ins), free ftty cids (FFA), cholesterol (C), nd phospholipid (PL). ANOVA, nlysis of vrince for probbility > F. A 3-fctor model ws used for initil nlysis of plsm nd liver prmeters. Further 2-fctor nlyses were performed in some cses. P vlues for differences between individul smples re given in the text. mrna LeBoeuj ClheIl, nd Kirk Nutritionl regultion of lipids nd polipoproteins in mice 123

4 (1, 5) using cdna probe (Gibco BRL, Githersburg, MD) for glycerol-3-phosphte dehydrogense (GAPDH) mrna s n internl stndrd. Sttisticl nlysis Results re reported s mens * SEM. Sttisticl differences were determined by multi-fctoril ANOVA using SSTAT for the Mcintosh (SSTAT, Inc., Evnston, IL). Post-hoc nlyses of significnce ws mde by Fisher's protected lest squres difference test. P <.5 ws ccepted s sttisticlly significnt. RESULTS Body weights were determined three times during this study for rodent chow- nd sffiower oil diet-fed mice. Initil body weights of BALB/c nd C57BL/6 mice were 19.6 k 1.3 g nd 18.2 k 1.2 g, respectively (men * stndrd devition) for the 3 mice of ech strin which were then rndomly distributed between diet groups. Within ech strin, finl body weights were nerly identicl for ll diet groups, suggesting tht the nimls were helthy nd te comprble clories. Finl body weights were 22.1 * 1.4 g for BALB/c mice nd 19. k 1. g for C57BL/6 mice. Body weights for mice fed the therogenic diet were not determined in this study, but mice hve been shown in mny preliminry experiments to either mintin or gin body weight (1-5% of initil body weight) over 2 weeks of feeding (R. LeBoeuf, unpublished dt). Finl body weights of mice used in the Triton studies re presented in Tble 2. Plsm lipid nd lipoprotein concentrtions Results of plsm lipid prmeters were exmined by three-wy ANOVA in terms of fsting time, mouse strin, nd diet. As shown in Tble 1, significnt 3-wy interctions were observed for plsm totl cholesterol (X), HDL cholesterol, nd plsm triglyceride concentrtions. A min effect on plsm totl cholesterol (IC) levels ws TABLE 2. Triglyceride (TG) secretion rtes following injection of Triton WR-1339" Time of Body TG Secretion Group Strin Fsting Weight Rte' h s ms/min A C57BLl f f.3 B C 5 7 B L * f.35 C BALBIc f f.92 D BALBIc * f.55 "Dt presented s men f SEM for 3 to 1 mice. 'Clculted s described in Mterils nd Methods. A two-w.y ANO- VA (strin versus fsting time) showed significnce only due to strin (F = 2.79, P <,2). Fisher's lest-significnt-difference test: A, C: P <.2; B, D; P <.55; A, B nd C, D: not significnt. dietry cholesterol (Fig. 1). Significnt increses in X vlues, regrdless of fsting time, were observed between mice of the sme strin when fed the therogenic diet (coco butter with cholesterol) s compred to rodent chow (P < O.OOl), nd when fed the sff/high (sfflower oil with cholesterol) s compred to sff/low (sfflower oil without cholesterol) diets (P <.1). Interction between fsting time nd diet ws reflected in decresed 7C levels t 16 h for C57BL/6 nd BALB/c mice fed the therogenic diet (P <.1) nd for C57BL/6 mice fed sff/high (P <.1). Significnt strin differences were seen for mice fed the therogenic diet (P <.1 t 4 h; P <.1 t 16 h), sff/low t 4 h (P < O.OOl), nd sff/high t 4 h (P <.5) nd 16 h (P <.1). HDL cholesterol levels were minly effected by diet nd strin (Tble 1). For mice fed sfflower oil diets, HDL cholesterol vlues (Fig. 1) were significntly lower upon ddition of dietry cholesterol t both fsting times for C57BL/6 (P <.1) nd BALB/c (P <.5 for 4 h; P <.1 for 16 h). BALB/c mice exhibited significntly higher HDL cholesterol levels for ll diets s compred to C57BL/6 mice (P <.5). The mount of plsm obtined from mice fed the therogenic diet ws not sufficient, due to technicl difficulties, to llow determintion of HDL cholesterol. In previous studies, HDL cholesterol vlues were 3 nd 6 mg/dl for therogenic diet-fed C57BL/6 nd BALB/c mice fsted overnight (2). Plsm triglyceride concentrtions were sensitive to ll effects except the interction of time nd strin (Tble 1). Thus, time effected similr chnges in plsm triglyceride levels for both strins. In prticulr, the 16 h fsting time ws ssocited with significnt increses in triglyceride levels (P <.4-.1) for ll but two cses (C57BL/6 fed therogenic; BALB/c fed sff/low). Diet lso provided min influence on plsm triglyceride levels which were lso strin- nd time-dependent. For instnce, triglyceride concentrtions for C57BL/6 mice were not significntly different t 4 h. At 16 h, n effect of diets contining cholesterol ws seen, s plsm triglyceride ws lower for mice fed the therogenic diet compred to rodent chow (P <.1) nd sff/high compred to sff/low (P <.1). In contrst, BALB/c mice fed high ft diets (therogenic nd sfflower oil) showed significntly lower triglyceride levels compred to BALB/c mice fed rodent chow t 4 h of fsting (P <.1). At 16 h, BALB/c mice showed n increse (P <.1) or no chnge in triglyceride levels with the ddition of dietry cholesterol. Thus, triglyceride responses were complex nd dependent upon combintion of nutritionl nd genetic fctors. Plsm glucose, insulin, nd free ftty cid (FFA) concentrtions A loss of tissue glucose stores, decrese in plsm insulin, nd n increse in plsm FFA re chrcteristics of semi-strvtion conditions (1, 11). Regrdless of diet or 124 Journl of Lipid Reserch Volume 35, 1994

5 mr Ir 4hFsted l 16hFsted CH Ath S- S+ & Ath S- S+ C57BU6 BALBlc - ci s- s+ Ci-I s- s+ C57BU6 C57BU6 BALWc BALWc strin, plsm glucose concentrtions were pproximtely 3% lower t 16 h s compred to 4 h of fsting (Fig. 2) (P <.1). Concomitnt chnges in mouse plsm insulin nd FFA concentrtions did not necessrily ccompny the glucose chnges nd were dependent upon strin nd diet. For instnce, significnt decreses in plsm insulin levels with fsting time were seen for BALB/c (P <.5-.1) but not C57BL/6 mice. Plsm insulin levels vried modestly (9-14 mu/ml) for C57BL/6 mice fed ll diets t both 4 nd 16 h fsting times. FFA levels incresed significntly for C57BL/6 mice fed ll diets upon fsting for 16 h s compred to 4 h (P <.5). BALB/c mice exhibited inconsistent chnges in FFAs with time nd diet. FFA levels were significntly higher t 16 h only for therogenic diet-fed BALB/c mice (P <.1), decresed for sff/low-fed mice (P < O.OOl), nd were unchnged for rodent chow- nd sfflhigh-fed mice. Differences between strins included significntly higher levels of insulin for BALB/c compred to C57BL/6 mice t 4 h (P <.1). In ddition, FFA levels were higher for BALB/c versus C57BL/6 mice (P <.1-.2) for ll but sfflower oil-fed mice fsted 16 h. Thus, glucose, insulin, nd FFA levels showed differentil effects due to fsting time, genetic, nd dietry fctors. Plsm polipoprotein (po) concentrtions Overll, plsm poa-i, A-11, nd A-IV concentrtions tended to remin the sme or decrese from 4 h to 16 h of fsting (Fig. 3). ApoA-I concentrtions showed effects from multiple interctions (Tble 1). Thus, diet nd time effects were strin-dependent. For instnce, t 4 h of fsting, poa-i levels for sfflower oil-fed C57BL/6 mice were higher thn observed for mice fed the rodent chow-bsed diets (P <.1). The poa-i levels for these sfflower oil-fed mice decresed significntly with incresed fsting time (P <.3). In contrst, poa-i levels for BALB/c mice remined reltively constnt for ll conditions t mg/dl. Significnt differences between strins were seen for 4 h-fsted mice fed rodent chow nd sfflower oil diets (P <.4). There were four min observtions concerning chnges in poa-i1 concentrtion. First, the primry effect of fsting time occurred for mice fed rodent chow, for which decreses were seen in both strins t 16 h (P <.4). Second, within ech fsting time group, mice fed rodent chow hd higher levels of poa-i1 thn mice fed the high ft diets for-strins BALB/c (P <.1) nd C57BL/6 (p <.3). Third, BALB/c mice showed greter concentrtions of poa-11 thn C57BL/6 mice (P <.2-.5) Fig. 1. Plsm lipid concentrtions in response to diet nd fsting time for C57BU6 nd BALB/C mice. Mice were mintined on either rodent chow (CH), diet contining rodent chow, 15 g/kg coco butter with 12.5 g% Ao'merol, nd 5 glkg sodium Or semi-synthetic for ll diets except sff/low t 4 h. Finlly, poa-ii levels diets contining 138 g/kg sfflower oil nd 5 g/kg sodium cholte without (s-! or with (s+) 5 g& cholesterol for 2 weeks. Animls were fsted were significntly decresed for C57BL/6 mice fed sff/high s compred to sff/low diets (P <.1) nd the therogenic versus rodent chow diets (P <.3), suggesting tht dietry cholesterol my influence plsm poa-i1 concentrtions in this strin. for either 4 h (solid br) or 16 h (htched br) nd bled from the orbitl sinus between 11 AM nd 1 PM. Plsm totl cholesterol, HDL cholesterol, nd triglyceride were determined s described under Mterils nd Methods. Ech br represents the men vlue * SEM obtined for 1 mice. Significnce levels re given in the text. LcBonrJ; Cldwell, nd Kirk Nutritionl regultion of lipids nd polipoproteins in mice 125

6 H 4hFsted 16hFsted M c1 9 4 ti" 1 J r; U 1 G CH Ath S- S+ CH Ath S- S+ C57BL16 BALBIc E v e 2.I M 4 H 4hFsted e 16hFsted loo CH Ath S- S+ CH Ath S- S+ C57BW6 BALBIc s.i J CI r - E v) 2 h d J 2 c4 - &l Ath S- S+ CH Ath S- S+ C57BW6 BALBlc " C% A& S- S+ Ci-l Ath S- S+ C57BW6 T BALBlc b 'U - $ 4 c1 m 4 3 E:.I J E -.I n -&I Ath S- S+ CH Ath S- S+ n v).- 4 C57BLJ6 T BALBlc -C%Ath S- S+ CH Ath S- k+ C57BU6 BALBlc Fig. 2. Plsm glucose, insulin, nd free ftty cid concentrtions in response to diet nd fsting time for C57BL/6 nd BALBlc mice. Experimentl conditions re s described in the legend to Fig. 1. Diets re rodent chow (CH), coco butter nd cholesterol (Ath), nd sower oil without (S-) or with (S+) cholesterol. Glucose nd free ftty cid determintions were done colorimetriclly nd insulin levels were determined by rdioimmunossy s described under Mterils nd Methods. Ech br represents the men vlue * SEM obtined for 3-1 nimls. Significnce levels re given in the text. Fig. 3. Plsm polipoprotein A-I, A-11, nd A-IV concentrtions in response to diet nd fsting time for C57BL/6 nd BALB/c mice. Apolipoprotein levels were determined by quntittive immunoblotting s described under Mterils nd Methods. Experimentl conditions re s described in the legend to Fig. 1. Diets re rodent chow (CH), coco butter nd cholesterol (Ath), nd sfflower oil without (S-) or with (S+) cholesterol. Ech br represents the men vlue f SEM obtined for 3-5 mice. Significnce levels re given in the text. 126 Journl of Lipid Reserch Volume 35, 1994

7 ApoA-IV concentrtions tended to be lower t 16 h versus 4 h, but reched significnce only for mice fed sfflower oil diets (P <.8). Within ech fsting time group, pd-iv concentrtions were the sme for C57BL/6 mice fed ll diets. In contrst, BALBlc mice fed the sff/low diet hd significntly higher poa-iv concentrtion thn BALB/c mice fed the other diets (P <.4). This diet ws the only one to result in significnt difference between strins t both fsting times (P <.1). n v).- U c s P.I c) t I 4 6 I 4hFsted 16 hfsted Heptic mrna levels GAPDH mrna ws used s our internl stndrd nd it ws not influenced by fsting time, diet, or mouse strins used here. This conclusion ws bsed on the signl intensity of GAPDH mrna bnds on utordiogrms which reflected the ethidium bromide stin intensity of 18s nd 28s bnds seen in gels prior to blotting. In ddition, mny of our smples were run repetedly in different combintions on seprte grose gels showing, in sideby-side comprisons, tht no trends in GAPDH mrna levels were observed. Although heptic poa-i mrna levels tended to increse with fsting time, only one of these vlues (BALB/c fed rodent chow; P <.5) reched sttisticl significnce due to lrge vritions (Fig. 4). Diet effects consisted of lower poa-i mrna vlues for BALB/c mice fed sfflower oil s compred to the chow nd therogenic diets (P <.1-.5). C57BL/6 poa-i mrna levels were not significntly different mong ll conditions. A diet-strin effect ws seen for mice fed the therogenic diet in which poa-i mrna levels were higher for BALB/c thn C57BL/6 mice (P <.6). ApoA-I1 mrna levels (Fig. 4) were influenced by time nd strin interctions. Bsed on 2-wy ANOVA nlyses, BALB/c showed significnt increses in poa-i1 mrna levels with time of fsting (P <.4). This ccounted for the higher levels of poa-i1 mrna levels seen for BALB/c versus C57BL/6 mice t 16 h (P <.3). Moderte diet effects were lso observed. At 16 h, poa-i1 mrna levels were reduced for C57BL/6 mice fed the high ft diets (therogenic nd sfflower oil diets) versus rodent chow, but sttisticl significnce ws obtined only for the sff/high diet (P <.6). ApoA-I1 mrna levels for BALB/c mice fed sfflower oil were lower thn for mice fed rodent chow nd therogenic diets. In fct, for BALB/c mice, chnges in poa-i1 mrna levels correlted to those of poa-i (* =.845; P <.1). ApoA-IV mrna levels (Fig. 4) were mrkedly responsive to fsting time (P <.1) with one exception (BALB/c mice fed the sff/low diet). ApoA-IV mrna levels incresed from 2- to 24-fold with fsting overnight s compred to 4 h. C57BL/6 mice showed no significnt differences in poa-iv mrna levels s function of diet t either fsting time. At 16 h, BALB/c mice fed the n." 2 c 1.- U CI t 4 6 "CH Ath S- S o.5 C57BU6 & Ath S- S+ BALWc n.n.. CH Ath S- S+ CH Ath S- S+ "I C57B6 BALWc CH Ath S- S+ CH Ath S- S+ C57B6 BALWc Fig. 4. Heptic levels of mrna specific for polipopmtein A-I, A-11, nd A-IV for C57BL/6 nd BALB/c mice. Experimentl conditions re s described in the legend to Fig. 1. Diets re rodent chow (CH), coco butter nd cholesterol (Ath), nd sfflower oil without (S-) or with (S+) cholesterol. Totl heptic RNA ws isolted nd 2 pg of ech preprtion ws subjected to Northern nlysis using S*P-lbeled cdna probes for the polipoproteins nd glycerldehyde-3-phosphte dehydrogense (GAPDH) s described under Mterils nd Methods. Autordiogrms were exmined by scnning densitometry to quntitte the reltive levels of the polipoprotein signls. Vlues shown re the men densitometric units for polipoprotein mrna normlized to levels of GAPDH + SEM for 3-5 mice. Significnce levels re given in the text. LeBoeuj Cidweii, nd Kirk Nutritionl regultion of lipids nd polipoproteins in mice 127

8 therogenic diet displyed significntly elevted poa-iv mrna levels s compred to mice fed the remining diets (P <.1-.17). Heptic lipid concentrtions The primry determinnt of heptic cholesterol concentrtion ws dietry cholesterol (Fig. 5). Thus, the therogenic nd sff/high diets resulted in over 5-fold increses in heptic totl cholesterol levels compred to rodent chow nd sff/low diets, respectively, for both strins (P <.1). There ws no effect of fsting time on heptic cholesterol levels within strins, nd strin effects were limited to the two diets contining dded cholesterol. C57BL/6 exhibited significntly higher levels of heptic cholesterol thn BALB/c mice (P <.1). Heptic triglyceride levels were mrkedly sensitive to fsting time nd were higher for 16 h versus 4 h fsted mice of both strins fed ll diets (P <.1-.11). Thus, elevtions in both heptic nd plsm triglyceride levels were observed with overnight fsting s compred to short-term fsting. Severl min effects were lso seen. For instnce, sff/high diets resulted in higher triglyceride levels s compred to sff/low diets for ll cses (P <.1). When contrsting rodent chow-to therogenic diet-fed mice, significnt decrese ws seen for C57BL/6 mice fsted for 16 h (P <.4), chnge not seen for BALB/c mice. Fsting time did not show min effect on heptic phospholipid levels. Phospholipid vlues were reltively constnt (12-18 mg/g tissue) for C57BL/6 nd BALB/c mice fed ll diets except the therogenic diet. Strin type ws min effect but yielded significnt differences between strins only for the therogenic diet (P <.1). VLDL production To ssess the contribution of triglyceride-rich lipoprotein production to the incresed plsm triglyceride levels t 16 h of fsting s compred to 4 h, plsm triglyceride levels were determined with time fter injection of mice with Triton WR Since plsm triglyceride levels incresed with fsting time regrdless of diet (Fig. l), mice fed rodent chow were used. C57BL/6 nd BALB/c mice fsted for 4 nd 16 h exhibited stedy increses in plsm triglyceride levels over the entire 12 min (regression nlysis vlues rnged from r2 = ). Plsm triglyceride vlues were significntly elevted bove those of control slineinjected mice t ech time point (dt not shown). The vlues t ech time point were used to estimte rtes of triglyceride secretion s described in Mterils nd Methods. As shown in Tble 2, triglyceride secretion rtes for C57BL/6 mice were the sme t 4 nd 16 h nd significntly lower thn vlues clculted for BALB/c mice t ech time point. Triglyceride secretion rtes t 4 nd h 9) 7 5 I.CI ; ;; c) M \ CI 3 2 u L v) n 4 hfsted 16hFsted " t% Ath S- S+ CH Ath s- s+ 12 r C57BLJ6 BALBIc " & Ath S- S+ 6 Ath s- s+ C57BL16 3 i.i 2 1 BALBIc n -. CH Ath S- S+ CH Ath S- S+ C57BL16 BALBIc Fig. 5. Heptic totl cholesterol, triglyceride, nd phospholipid concentrtions in response to diet nd fsting time for C57BL/6 nd BALB/c mice. Experimentl conditions re s described in the legend to Fig. 1. Diets re rodent chow (CH), coco butter nd cholesterol (Ath), nd sfflower oil without (S-) or with (S+) cholesterol. Lipids were extrcted ccording to Folch et l. (18) nd lipid determintions were done using spectrophotometric nlyses s described under Mterils nd Methods. Ech br represents the men vlue * SEM obtined for 4-5 mice. Significnce levels re given in the text. 128 Journl of Lipid Reserch Volume 35, 1994

9 16 h observed for BALB/c were not significntly different. Thus, genetic fctors ppered to be the min influence on rtes of plsm triglyceride secretion in these mice. DISCUSSION This report demonstrtes tht spects of lipoprotein trnsport re ltered by nutritionl sttus in mice. For nutritionl studies, it is helpful to fst nimls prior to collection of blood ndlor tissues to void fluctutions due to postprndil events. Our results show tht durtion of fsting hs strong influence on mny metbolic prmeters in mice. The primry effect of fsting for 16 h s compred to 4 h ws to decrese plsm glucose levels, elevte triglyceride levels in the plsm nd liver, nd increse the mount of heptic poa-iv mrna. These chnges were generlized phenomen s they occurred regdless of strin nd diet. Other fetures of incresed fsting were modest chnges in levels of plsm totl nd HDL cholesterol, nd polipoproteins, nd in liver poa-i nd poa-i1 mrna. Vritions in these prmeters were lso seen due to diet nd genetic bckground. Finlly, chnges in plsm insulin nd FFA concentrtions were differentilly sensitive to fsting time, strin, nd diet. In this study, two ctegories of diets were used. The first ctegory included two diets previously shown to differentite therosclerosis susceptibility mong mouse strins (8). These diets consisted of either chow fed lone or mixed with coco butter, cholesterol nd bile cid. The second ctegory included two semi-synthetic diets developed to systemticlly identify nd chrcterize genes susceptible to mnipultion by dietry cholesterol nd polyunsturted ftty cids (17). These diets represent two ctegories used by us (8, 17) nd others (3, 28) for studies of mouse lipid trnsport nd differ mrkedly in mount nd type of mny components, including protein, fiber, bile cid, nd fts. et, mjor chnges in plsm lipids nd expression of t lest one polipoprotein were observed regrdless of diet, suggesting tht extent of food restriction needs to be tken into considertion in describing mouse phenotypes. A mjor finding ws tht fsting for 16 h resulted in elevted plsm triglyceride levels compred to fsting for 4 h. In humns (29 nd refs. therein) nd rts, overnight fsting leds to decresed plsm triglyceride levels. However, reports conflict s to the effect of fsting on plsm triglyceride concentrtions in rodents. Triglycerides hve been reported either to increse (1, 3, 31) or decrese (14, 32, 33) during fsting. The contrdictory reports my be prtilly explined by the extent of fsting, s decresed plsm triglyceride levels were found in studies using severe strvtion (> 24 h) conditions. The Triton WR-1339 studies showed tht VLDL production ws not ltered by fsting. Thus, the incresed triglyceride levels in plsm t 16 h must be due to decresed VLDL clernce. The reduced clernce of VLDL t 16 h ws likely due to decresed lipolysis by LPL (5). Lipolysis of triglyceride is necessry step for efficient ctbolism of VLDL (34) nd chylomicrons (35). Consistent with this is the observtion tht, in rts, LPL ctivity chnges rpidly in response to chnges in nutritionl sttus (36). Interestingly, min determinnt of triglyceride secretion rte ws strin type with BALBk mice exhibiting significntly greter triglyceride secretion rtes thn C57BL/6 mice. Heptic triglyceride levels were lso elevted t 16 h versus 4 h of fsting. During strvtion, de novo ftty cid synthesis is decresed (37, 38) nd heptic triglyceride is derived primrily from esterifiction of glycerol with preformed exogenous ftty cids (39). In fct, in vivo studies suggest tht incresed vilbility of circulting FFAs nd glycerol stimulte triglyceride synthesis in the liver (39). This my hve occurred in our mice, s elevtions in plsm free ftty cids with incresed fsting time were observed, t lest for C57BL/6 mice, nd fsted mice re known to exhibit high levels of free glycerol (3-4 mgldl) (1, 4). Thus, compred to the 4 h-fsted mice, our 16 h-fsted mice were probbly synthesizing more triglycerides from peripherlly derived glycerol nd FFAs, nd more of these triglycerides were retined rther thn secreted from the liver. Severl reports suggest tht VLDL secretion is dependent upon the vilbility of pob (12, 39) s well s lipid components. Thus, one explntion for lck of VLDL production in our mice t 16 h ws low pob protein synthesis upon overnight fsting, which is currently being tested. Another mjor effect of fsting ws on heptic poa-iv mrna levels, which were mrkedly elevted t 16 h compred to 4 h. It is tempting to speculte tht the coincidentl increses in heptic poa-iv mrna nd triglyceride levels suggest role for poa-iv in intrcellulr triglyceride storge, pckging into lipoproteins, ndlor secretion. Such roles for poa-iv in the intestine hve been suggested bsed on elevtions in both poa-iv mrna nd protein levels in response to ft feeding (7, 41). Expression of poa-iv in the liver, but not intestine, hs repetedly been shown to be sensitive to hormonl s well s dietry controls (42, 43). For instnce, tretment of rts with thyroid hormone (42, 44), corticosteroids (43, 45), or dietry sucrose (46) elevtes heptic poa-iv mrna levels, while hypothyroidism induced by PTU (42, 44, 47), estrogen (42, 48, 49), nd clofibrte (42) reduces heptic pd-iv mrna. Compring these nd other studies of heptic poa-iv expression to mesurements of heptic triglyceride ccumultion nd secretion (Tble 3) suggests tht conditions tht increse liver LCBocuj Cldwell, nd Kirk Nutritionl mgultion of lipidti nd polipoproteine in mice 129

10 TABLE 3. Reltionships between heptic poa-iv mrna, nd heptic triglyceride (TG) concentrtion nd VLDL secretion s function of fsting, hormone, nd dietry tretment ApoA-IV mrna TG Accumultion VLDL Secretion Tretment Effects Ref. Effects Ref. Effects Ref. Fsting < 48 h Hyperthyroid Dietry sucrose Corticosteroids Dexmethzone H ydrocortizone Insulin Fsting > 48 h PTU Estrogens Fibrtes t here, 15 t 15, 42, 44, t 46 t 43, 45, 56 t 42, 45 t , , 44, , t t 55 t t 61 t , , t, 1 3, 31, 37 t 44, 5 t 55 t 45, 56, 58-6 t , 57, , 33, , 5 t 6, , 67 Effects of tretments compred to control vlues is noted s n increse (t), decrese (l), or no chnge (---), s described within the listed references triglyceride ccumultion lso increse poa-iv mrna. However, ccumultion but not secretion ws seen for estrogen tretment nd short-term fsting ( < 48 h) (5), s lso shown in this study, suggesting n uncoupling between triglyceride ccumultion nd secretion. Thus, we hypothesize tht in the liver of rodent nd perhps vin species, poa-iv is needed for storge of triglyceride nd/or pckging into VLDL, but tht incresed expression of poa-iv does not led to enhnced secretion of VLDL. Recently, Reue et l. (51) showed tht both trnscription nd turnover contribute to stedy-stte heptic poa- IV mrna concentrtion. Thus, elevtions in poa-iv mrna levels with fsting time were likely due to combintion of these events. Reue et l. (51) lso showed tht the expression of poa-iv in liver contributes to plsm concentrtions of poa-iv protein in mice. In contrst to heptic mrna levels, poa-iv plsm protein levels were lower for mice fsted 16 h versus 4 h of fsting. Since plsm poa-iv is derived from both heptic nd intestinl sources (43), the decrese in plsm poa-iv levels for 16 h-fsted mice probbly reflected blnce between ctbolism of liver-derived plsm poa-iv nd diminished contributions from intestinl synthesis. Although our reltive mrna vlues for poa-i nd A- I1 for 16 h-fsted C57BL/6 nd BALB/c fed chow nd therogenic diets were comprble to those of Willims et l. (7), the poa-iv mrna vlues were quite different. For instnce, Willims et l. (7) observed nerly 4-fold increse in poa-iv mrna upon feeding C57BL/6 mice the therogenic diet compred to mice fed rodent chow. Chow fed BALB/c mice exhibited nerly %fold higher poa-iv mrna levels s compred to C57BL/6 mice nd 2-fold reduced levels upon feeding therogenic diet. Our work showed mrked increse in heptic poa-iv mrna levels t 16 h versus 4 h of fsting for both strins fed ll diets. In contrst, poa-i nd A-I1 mrna levels were less ffected by fsting time. Our results imply tht fsting time ffects the regultion of poa-iv in generlized wy (not dependent upon diet or genetic bckground). Discrepncies with mrna levels reported by Willims et l. (7) were lso described by Reue et l. (51) who ttributed the differences to non-genetic fctors. We suggest tht such non-genetic fctors influencing heptic levels of poa-iv mrna re differences in the exct times of fsting nd perhps time of dy (diurnl cycle) during which tissues were collected. Cholesterol levels in plsm were higher for mice fed diets with dded cholesterol compred to the compnion diets without dded cholesterol, s observed previously (17, 2). Plsm HDL cholesterol concentrtions were lso influenced by the diets, prticulrly for the C57BL/6 mice. Severl reports hve shown tht C57BL/6 mice fed diets rich in cholesterol, sturted fts, nd bile cids exhibit mrked decreses in HDL cholesterol levels nd tht this effect is subdued in other strins such s BALB/c nd C3H/He (2, 8). The decrese in C57BL/6 HDL levels is medited by incresed HDL ctbolism controlled, t lest in prt, by the gene Ath-I (8). The dietry component responsible for this effect is unknown. Since C57BL/6 mice hve shown the decrese in HDL cholesterol with sturted ft-(2, 8, 28, 52), polyunsturted ft- (Fig. l), nd low ft- (52) bsed diets, the primry dietry component responsible for decresed HDL levels is likely to be cholesterol. In support of this ide is the observtion tht high levels of dietry ft fed to C57BL/6 mice in the bsence of cholesterol result in the mintennce (Fig. 1) or elevtion (52) of plsm HDL cholesterol levels in comprison to mice fed low-ft diets. Decreses hve been observed in HDL levels for C57BL/6 mice fter cholesterol feeding tht re ccompnied by decreses in polipoprotein components. In prticulr, poa-i nd poa-i1 levels decrese by pproximtely 3% nd 5%, respectively, compred to levels seen for mice fed control diets (2, 8). Our C57BL/6 mice 13 Journl of Lipid Reserch Volume 35, 1994

11 did not show the chrcteristic drop in plsm poa-i levels upon consuming the therogenic diet. Perhps longer feeding times (> 2 wks) re required to cuse more extensive loss of plsm poa-i. In contrst to poa- I, poa-i1 plsm levels decresed s much s 2-fold for both C57BLJ6 nd BALB/c mice fed ny of the high-ft diets compred to levels for mice fed chow within ech time point. At 16 h of fsting, the decreses were gretest for C57BL/6 mice fed the therogenic nd sff/high diets suggesting tht both the high ft nd cholesterol dietry components contributed to loss of poa-11. C57BL/6 nd BALB/c mice vried in the response of insulin levels to fsting times. Insulin levels contribute to the regultion of t lest two lipolytic enzymes, lipoprotein lipse nd hormone-sensitive lipse (53). In dipose tissue, lipoprotein lipse nd hormone sensitive lipse show reciprocl regultion by insulin. Thus, under conditions of strvtion, lipolysis of plsm triglycerides decreses nd lipolysis of triglycerides stored in dipose tissue ccelertes. Consistent with these expecttions, C57BL/6 mice exhibited diet-independent decreses in glucose nd elevtions in plsm concentrtions of triglyceride nd FFA. Surprisingly, concomitnt decreses in insulin levels were not seen, suggesting tht the role of insulin in lipse ctivities my be loclly medited or tht lipse ctivities re ltered by fctors other thn or in ddition to insulin in this strin. In contrst, BALB/c mice exhibited decreses in both glucose nd insulin levels. Although levels of plsm FFAs tended to be higher in BALB/c thn C57BL/6 mice fed the sme diets, overt chnges in FFA levels due to fsting time were irregulr nd dependent upon diet in BALB/c mice. This indictes different blnce between tissue lipolysis nd heptic uptke of plsm FFA thn seen for C57BL/6 mice. Tht genetic differences exist mong strins with respect to insulin sensitivity hs been demonstrted by Surwit et l. (54) who disssocited fsting glucose levels from insulin sensitivity mong progeny of C57BL/6 nd A/J mouse strins. Thus, incresed fsting resulted in elevtions in plsm triglyceride concentrtions, but concomitnt chnges in plsm insulin nd FFA concentrtions were strin-dependent. In summry, durtion of fsting lters mny metbolic prmeters used to chrcterize the lipid trnsport system in mice. In ddition, the mgnitude of these ltertions is influenced by dietry nd genetic fctors. Mice re prticulrly sensitive to food restriction-induced depletion of their lipid reserves becuse of their high overll metbolic rte even s compred to rts (11). Significnt chnges in ptterns of fuel utiliztion occur within 3 h (1) fter feeding tht re reflected in chnges in lipid metbolism. Thus, we suggest tht 4 h s opposed to overnight fsting is preferble prior to studies of the lipid trnsport system in mice. I We re grteful to Dvid IAlessio (University of Wshington, Settle, WA) for the plsm insulin nd glucose mesurements, to John Lernmrk nd Deborh Wilson for technicl ssistnce in lipid determintions, to Gile Moe (University of Wshington) for sttisticl support, nd to Kren Reue (Wdsworth VA Medicl Center nd University of Cliforni, Los Angeles, CA), John F. Orm (University of Wshington), nd John Brunzell (University of Wshington) for their suggestions concerning this mnuscript. This work ws funded by NIH Grnt #HL42333 nd the Ntionl Diry Promotion nd Reserch Bord nd dministered in coopertion with the Ntionl Diry Council. Mnuscript rcccivcd 24 My 1993 nd in reuiscdfoim 31 August REFERENCES 1. Doolittle, M. H., R. C. LeBoeuf, C. H. Wrden, L. M. Bee, nd A. J. Lusis A polymorphism ffecting polipoprotein A-I1 trnsltionl efficiency determines high density lipoprotein size nd composition. J. Biol. Chem. 265: Ishid, B.., P. J. Blnche, A. V. Nichols, M. shr, nd B. 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