Proc. Natl Acad. Sci. USAVol. 80, pp. 2179-2183, April 1983Biochemistry

Quantitative correlation between proteolysis and macro- andmicroautophagy in mouse hepatocytes during starvationand refeeding

(liver/lysosomes/electron microscopy/protein synthesis/cytoplasmic growth)

GLENN E. MORTIMORE, NANCY J. HUTSON*, AND CYNTHIA A. SURMACZDepartment of Physiology, The Milton S. Hershey Medical Center, The Pennsylvania State University, Hershey, Pennsylvania 17033

Communicated by Charles R. Park, January 4, 1983

ABSTRACT Cytoplasmic protein in hepatocytes is seques-tered and degraded by two general classes of lysosomes, overt au-tophagic vacuoles (macroautophagy) and dense bodies (microau-tophagy). Volumes of the apparent space in each class that containthe internalized protein, together with estimates of cytoplasmicprotein concentration, were used as a basis for predicting rates ofprotein degradation by the lysosomal system in livers of fed, 48-hr starved, and starved-refed mice. Assuming that the turnoverof all sequestered protein is equal to that previously determinedin overt autophagic vacuoles (0.087 min'), we obtained closeagreement between predicted and observed rates in the threeconditions studied. The two autophagic components, though, ex-hibited different patterns of regulation. Microautophagy followeda downward course through starvation and into refeeding, a trendthat explained fully the fall in absolute rates of protein degra-dation during starvation. By contrast, macroautophagy remainedconstant throughout starvation but was virtually abolished withrefeeding. Whereas regulation of the latter can be explained largelyby immediate responses to the supply of amino acids, present evi-dence together with results of others indicate that microseques-tration could be linked to functional and quantitative alterationsin the smooth endoplasmic reticulum. Both types of regulationcontributed equally to the marked suppression of proteolysis dur-ing cytoplasmic regrowth.

Protein and other cytoplasmic constituents in hepatocytes areinternalized within the lysosomal system in two general ways:(i) by the formation of overt autophagic vacuoles, a highly vis-ible mechanism involving the sequestration of organelles withinsmooth-surfaced membranes (1-5), and (ii) by the engulfmentof small bits of cytoplasm that subsequently appear in densebodies (1, 3-5), a microautophagic process that we believe ac-counts for basal protein turnover (5). Although both are ongoingfunctions, we have shown in perfused livers of fed animals thatthe addition or deletion of amino acids can regulate overt au-tophagy rapidly over a wide range without appreciably affectingmicroautophagy (3, 5). On the other hand, the induction of cy-toplasmic growth, such as that produced by starvation and re-feeding, is associated with decreases in rates of protein break-down (6, 7) and sequestered protein (7) to levels that can beexplained only by the suppression of both autophagic functions.Nothing is presently known, however, of the way microinter-nalization is regulated.The present investigation is an attempt to quantify by stereo-

logic methods the contributions of each process to proteinbreakdown in livers of fed, starved, and starved-refed mice andto compare degradation rates predicted by this approach withexperimentally determined rates. Close agreement was found

under all conditions, although activities of the individual au-tophagic components were affected in distinctly independentways. Rates of overt autophagy were interpretable from thegeneral state of amino acid deprivation, whereas changes in mi-croautophagy appeared to relate to intrinsic alterations withinthe cell.

EXPERIMENTAL PROCEDURESAnimals. Male, 7-week-old CD-1 mice (Charles River Breed-

ing Laboratories), initially weighing 33-35 g, were used in allexperiments. Except during periods of food withdrawal, theyhad free access to water and Charles River chow and were main-tained in an environmentally controlled room (light on 0700 to1900). Starvation was induced by removing food at 0900, a pointthat served as a timing reference for all subsequent procedures.To minimize variability, large numbers of animals were em-ployed for the determination of changes in liver protein contentduring starvation and refeeding. At intervals, groups of micewere killed by decapitation and the livers were removed for thedetermination of protein (7, 8). Liver volumes were measuredby saline displacement and densities were computed from theweights. Livers were also fixed by vascular perfusion and thinsections were prepared for electron microscopy as described (3,5, 9); standard stereologic procedures were employed for mea-suring volume densities of lysosomal components (3, 5, 9).

Determination of Hepatic Protein Synthesis and Degra-dation. Rates of protein synthesis in liver were measured in sep-arate groups of mice from the incorporation of L-[U-'4C]valine(295 mCi/mmol; 1 Ci = 3.7 x 1010 Bq; New England Nuclear)during 2-hr cyclic perfusions. The details of this method as ameans for determining resident protein synthesis in vivo havebeen fully described and validated in a recent paper (7). Ratesof resident protein breakdown were calculated by subtractingthe average rates of change of total liver protein from corre-sponding rates of synthesis (7).

Liver Volumes. Liver parenchymal volume and its majorfractions were determined in order to provide a basis for com-puting absolute lysosomal volumes (Table 2) and concentrationsof hepatocyte protein (Table 3). The results and description ofthe methods used, except those for extracellular volume, aregiven in Table 1 and the preceding text. For convenience ofexplanation, extracellular volume may be divided into threecomponents: (i) a nonvascular fraction, composing 0.05 of totalliver volume (10), 92% of which is continuous with the plasmacompartment (11); (ii) a residual blood space of 0.03 (10); and

Abbreviations: ER, endoplasmic reticulum; DB, dense bodies; AV, au-tophagic vacuoles.* Present address: Central Research, Pfizer Inc., Eastern Point Rd.,Groton, CT 06340.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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(iii) an additional quantity of blood that is retained when ani-mals, as in this study, are not exhaustively exsanguinated (10).Williams and Woodbury have shown that the chloride space ofthis extra blood is directly related to the difference between theobserved extracellular chloride space and a basal value of 0.20,determined in exsanguinated rats (10). With their method, weobtained differences (mean ± SEM) of 0.104 ± 0.009 and 0.102± 0.014 in seven and nine fed and 48-hr starved mice, re-spectively. Results were higher in 24-hr refed mice (0.182 +0.024, n = 8), but the distribution of [14C]inulin remained un-changed. Because intracellular chloride might have increasedduring rapid cytoplasmic regrowth, we used the average dif-ference in fed and starved animals (0.103) for all groups. Thevolume of this extra space was computed from the expression

[ (1 - 0.54) 1A (PCV)- + 1],

in which A is the above chloride space difference and 0.54 isthe nominal chloride space of erythrocytes (12). Average he-matocrits (PCV, packed cell volume) for the four groups in theorder listed in Table 1 (left to right) were 0.365, 0.466, 0.425,and 0.386. Values of total extracellular space in line E of Table1 are the sums of i, ii, and iii above, expressed as fractions ofliver parenchymal volume.

In tissue fixed for electron microscopy, the fractional distri-bution of cellular components would be slightly increased ow-ing to the washout of sinusoidal blood and the consequent re-duction in extracellular volume. Values of hepatocyte cytoplasmin Table 1 (line C, parentheses) were computed from a reportedextracellular space of 0.159 (11).

Presentation of Data. Values expressed on the basis of totalliver were normalized to an initial body weight of 34 g. Wherepossible, results are presented as means ± SEM with numbersof observations in parentheses.

Table 1. Volume of liver parenchyma and its distribution in fed,starved, and starved-refed mice

Starved Refed RefedVolume Fed 48 hr 12 hr 24 hr

Total volume, ml/34-g mouseLiver 1.670 1.039 1.843 1.771Parenchyma 1.577 0.946 1.750 1.678

Fraction of parenchymal volumeA. Hepatocytes 0.727 0.679 0.727 0.729B. Hepatocyte nuclei 0.071 0.071 0.071 0.071C. Hepatocyte cytoplasm 0.656 0.608 0.656 0.658

(0.715) (0.678) (0.715) (0.718)D. Sinusoidal cells 0.045 0.075 0.041 0.042E. Extracellular 0.228 0.246 0.232 0.229

Liver volumes for the groups above (left to right) were calculated bydividing weights in Fig. 1 by separately measured densities, which were,respectively: 1.126 ± 0.007, 1.068 ± 0.024,1.145 ± 0.009, and 1.135 +0.019 g/ml (n = 6-8). Liver parenchyma (liver minus connective tis-sue, large vessels, etc.) makes up 92.4% of liver volume in 24-hr starvedrats (11). Because -27% of liver volume in mice and rats is normallylost in 24 hr of starvation, the volume of supporting tissue would beabout 0.093 ml/34-g mouse, a value assumed to remain fixed duringstarvation and refeeding. The volume of sinusoidal cells was also as-sumed to remain constant and, from Blouin et al. (11), was computedto be 0.086 ml/34-g mouse. Hepatocyte nuclear volume has been shownto decrease in proportion to liver volume during starvation (13); the dis-tribution below was based on data from ref. 11. Values in lines A, D, andE total 1.0; A and C were determined by difference. The distributionsin parentheses (line C) were computed for fixed tissue (see text).

RESULTS AND DISCUSSIONAlterations in Liver Protein Content and in Rates of Protein

Synthesis and Degradation During Starvation and Refeeding.The results in Fig. 1 are in general agreement with our earliermeasurements of responses to starvation and refeeding in themouse (7). A closer approximation in this study of early changesin liver protein indicates that the initial rate ofdepletion is greaterthan that at 48 hr, an observation that may relate to the morerapid decline in liver weight in the first 24 hr (7). The 36% over-all loss of liver protein during 48 hr of starvation was almostcompletely regained in the following 24-hr period of food in-take. As emphasized in previous investigations (6, 7), proteinregrowth was dominated by a dramatic down-regulation of pro-tein degradation to rates approximately 10% of fed control val-ues (7). While total protein synthesis per liver returned to pre-starvation levels, it is evident that the observed suppression ofproteolysis would have caused some cytoplasmic growth evenif synthesis had not changed.

Volumes of Lysosoma-Vacuolar Components. In earlierstudies (5, 14), we found close agreement between predictedand observed values of cytoplasmic protein internalized by ly-

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FIG. 1. Alterations in the content of liver protein (A) and in ratesof protein synthesis and degradation (B) during starvation and re-feeding in the mouse. Net rates of liver protein loss at 0 and 48 hr werecalculatedby least-squares regression from the first three and last fourpoints; rates of gain at 12 and 24 hr of refeeding represent the meanrate between 60 and 72 hr. Rates of resident protein synthesis, deter-mined as previously detailed from the 2-hr incorporation of L-[U-14C]valine during cyclic perfusion (7), were 3.73 ± 0.26, 3.96 ± 0.20,3.56 ± 0.21, and 3.91 ± 0.16 mg hr-1/g of liver, respectively, in fed,48-hr starved, 12-hr refed, and 24-hr refed mice (four per group). Ratesper liver were obtained by multiplying these values by the followingaverage liver weights (g) for the same groups: 1.88 ± 0.03, 1.11 ± 0.03,2.11 ± 0.07, and2.01 + 0.04 (8-12pergroup). Proteindegradationthenwas computed as the difference between synthesis and net rates of pro-tein change.

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-sosomes in perfused livers of fed rats when we used the ag-gregate volumes of AVd (degradative autophagic vacuoles) +total DB (dense bodies) (for description, see legend to Table 2)in the calculations. Because these were acute studies in whichovert autophagy but not the total volume ofDB was altered, wecould draw few conclusions concerning the nature of microau-tophagy or its regulation. Two relevant points, however, didemerge from experiments in which macroautophagy was vir-tually eliminated by amino acid additions (5, 14). First, the per-sistence of type A DB (for description, see legend to Table 2)clearly suggested that microautophagy is an ongoing functioneven when macroautophagy is suppressed. Second, the appar-ent space of sequestered protein was shown to approximate thetotal volume of DB. Although the observed volume-of type ADB constitutes =30% of the total DB in livers of fed animals(3, 5, 9), it must be recognized that this percentage greatlyunderestimates their actual volume. This is so mainly becausethe electron-lucent zone that, marks these particles is consid-erably smaller than the surrounding electron-dense region (seebelow) and would not be visualized in the majority of randomsections. But how the sequestered protein is distributed withinthe population of DB or a given component of it is not at allclear. Because space would be required for products of diges-tion and intrinsic lysosomal proteins, it follows from the secondpoint above that the concentration of internalized protein mustbe higher than that in cytoplasm. This is not an unreasonablepossibility, because in the process of formation of AVi (initialautophagic vacuoles), cytoplasm is believed to lose as much ashalf of its water content immediately after sequestration (5).

In contrast to the above amino acid effects, which were largelyrestricted to macroautophagy (5, 14), longer-termed metabolicalterations induced changes in both micro- and macroauto-phagic components. As shown in Table 2, absolute volumes oftotal DB decreased progressively during starvation and refeed-ing. In relative terms, the volume of type A DB fell to a greaterextent than did total DB, decreasing 79% as compared with 49%for the total by 12 hr of refeeding. Similarly, the volume ratioof type A to R fell by nearly 50% after 48 hr of starvation, ad-vancing to 67% in the initial periods of refeeding. This apparentshift in the distribution of the two classes of particles suggests

that microautophagy was selectively reduced. We consideredthe possibility that only the electron-lucent zones had becomesmaller. However, no differences were found in zone-to-par-ticle volume ratios, which averaged 0.14, 0.13, and 0.14, re-spectively, in the fed, 48-hr starved, and starved-refed (12 +24 hr) groups. From these considerations as well as the generalappearance of the particles, we conclude that the overall struc-ture of type A lysosomes was not affected.

Macroautophagy followed a distinctly different course (Table2). In general agreemenrwith others (15, 16), absolute volumesof AVd remained comparatively constant during starvation butdecreased by more than 90% after refeeding. We should per-haps point out that the doubling of the fractional volume of totalAV by 48 hr of starvation noted in Table 2 was not an intrinsiclysosomal alteration, but rather the consequence of a reductionin cytoplasmic volume.

Uptake and Degradation of Cytoplasmic Protein by Lyso-somes. In predicting the quantities of protein sequestered bydense bodies during starvation and refeeding, we consideredtwo possibilities for computing the hypothetical protein spaceand its decrease. The first was the observed volume of DB it-self. The second, designated DBp, was the volume of DB in fedanimals, multiplied by the relative volume of type A particlesin each state. We reasoned that if type A lysosomes are in factmarkers for microautophagy, the apparent space should de-crease in proportion to the fall in type A particles seen in Table2. Calculated values for DBp are given in the legend to Fig. 2.They differ from absolute volumes of DB in Table 2 only instarved and starved-refed states, in which the numbers reflectthe shift in DB distribution from type A to type R.

Quantitative predictions of internalized protein, based onabsolute volumes of DBp + AVd and averages of minimal andmaximal estimates of cytoplasmic protein concentration in Ta-ble 3, were in fact remarkably similar to experimentally de-termined values of degradable intralysosomal protein in liverhomogenates reported earlier (7). The latter results, measuredin control fed and 48-hr starved then 12-hr refed mice (7) andcorrected for a known underestimation of -10% (14), were 1.89and 0.24 mg of protein per liver (34-g mouse). Predicted values,computed from DBp + AVd as described above, were 2.01 and

Table 2. Lysosomal volumes in hepatocytes of fed, starved, and starved-refed mice

Volumes Fed Starved 48 hr Refed 12 hr Refed 24 hrFractional volumes, ,Al/ml of cytoplasm

AVi + AVd 1.55 ± 0.58 3.01 ± 0.62 0.10 ± 0.09*11 0.13 ± 0.07*¶Type A DB 1.18 ± 0.26 0.98 ± 0.27 0.22 ± 0.03tt 0.30 ± 0.15*Type R DB 3.18 ± 0.99 4.94 ± 0.82 1.80 ± 0.35§ 1.38 ± 0.43§

Absolute volumes, /ul/liver of 34-g mouseAVd 0.839 (100) 0.927 (10) 0.060 (7) 0.075 (9)Total DB 4.916 (100) 3.797 (77) 2.528 (51) 2.024 (41)Type A DB 1.331 (100) 0.629 (47) 0.275 (21) 0.361 (27)Type R DB 3.585 (100) 3.168 (88) 2.253 (63) 1.663 (46)

AVi and AVd denote initial (sequestrational) and degradative forms of overt or macroautophagy (5, 9),with AVd making up -48% of AVi + AVd over the full range of regulation (5). After a lag of 7-8 min,AVi are transformed into AVd, at which time intravacuolar digestion begins (5). DB consist of all re-maining lysosomes (1-5). On the average they are smaller than AVd and can readily be divided into twogroups according to whether the profiles contain a sharply demarcated, electron-lucent zone (type A) ornot (type R). Because these zones are usually filled with glycogen or other granular material, togetherwith occasional membrane remnants (3, 5, 9), type A DB may be considered autophagic. Some may containendocytosed material, but the effect on volume measurements is probably negligible. Fractional cyto-plasmic volumes were converted to absolute volumes by multiplying the values by fractional parenchymalvolumes of hepatocyte cytoplasm in livers fixed for electron microscopy (Experimental Procedures) andby corresponding parenchymal volumes (Table 1). Each group consisted of three to five livers. Numbersin parentheses are percentages of fed control values. Probabilities that differences could have arisen bychance are as follows. Versus fed: *, P < 0.05; t, P < 0.01; versus starved: t, P < 0.05; §, P < 0.02; ¶, P

2182 Biochemistry: Mortimore et al.

Table 3. Distribution of protein in livers of fed, starved, andstarved-refed mice

Starved Refed RefedProtein Fed 48 hr 12 hr 24 hr

Liver protein, mg/ml of parenchymaA. Total 274.6 291.8 190.3 244.4B. Hepatocyte 240.7 248.5 152.9 209.5

Hepatocyte protein, mg/mlC. Per cell volume 331.1 366.0 210.3 287.4D. Per cytoplasmic

volume 366.9 408.7 233.1 318.4

Results in line A were determined by multiplying observed liver pro-tein by the ratios of mean liver weight to parenchymal volume (Fig. 1and Table 1). Protein concentrations (mg/g) in the four groups were(left to right) 230.3 ± 6.5, 248.7 ± 4.0, 157.8 ± 2.7, and 204.0 ± 3.1 (8-12 livers per group). Line B.then was obtained by subtracting from Athe following sources of nonhepatocyte protein: (i) sinusoidal cell pro-tein, estimated as -45 mg/ml of parenchyma (17, 18), and (ii) extra-cellular protein, computed from the content of erythrocytes and com-bined plasma and nonvascular spaces (see Experimental Procedures).Erythrocyte protein was taken as 336 mg/ml of packed cells [280 mgof hemoglobin (personal communication from Richard Lee, Charles RiverBreeding Laboratories), plus a 20% allowance for other proteins]; pro-tein in the noncellular space was assumed to be similar to that of plasma,64 mg/ml. Total nonhepatocyte protein then was calculated from theparenchymal distributions of these components. Lines C and D rep-resent two estimates of hepatocyte protein concentration made by di-viding line B by appropriate distributions in lines A or C of Table 1. Cis minimal and assumes a uniform distribution of protein in cell water.However, because the nucleus is very likely lower in protein then cy-toplasm, a maximal estimate D was computed on the basis that proteinwas distributed only in cytoplasmic water;

0.24 mg, respectively. By contrast, the use of total DB as anestimator of the protein space gave a value for the starved-refedrat livers that was 0.59 mg-2.5 times higher than the ob-served.

Having tested the predictability of internalized protein, weextended. our calculations to proteolytic rates. For this we as-sumed that the turnover constant of the sequestered protein isthe same for microautophagic components as we have dem-onstrated in macroautophagy, 0.087 min- (5). Several lines ofevidence support this assertion (5,. 7, 14), but the major ar-gument rests on the direct proportionality that has been dem-onstrated between degradable intralysosomal protein and ratesof protein degradation over 95% of the proteolytic range frommaximum to slightly above zero (7). While this relationship doesnot, of itself, exclude other possible proteolytic mechanisms(14), it does strongly indicate that the velocity of degradationof internalized protein is a linear function of the quantity pres-ent (7, 14).

Fig. 2 shows that relative rates of resident protein degra-dation predicted from AVd + DBp and the above rate constantwere virtually identical to observed values up to 60 hr. whereaspredictions based on AVd + total DB deviated from them ap-preciably. The apparent discrepancy noted at the 72-hr pointwill be discussed below. Relative agreement was not affectedby more than ±4% when maximal and minimal rather than av-erage estimates of cytoplasmic protein were employed in thecalculations. As shown in the legend to Fig. 2, the predictedabsolute rate of protein degradation in fed animals averaged92% of the observed (88% minimal, 96% maximal).

Regulation of Microautophagy. Whereas overt autophagy issubject to direct regulation by amino acids, insulin, and glu-cagon (19), microautophagy is not (5, 20). What emerged un-expectedly then was the pronounced decline in this processduring starvation and refeeding (Table 2). The fact that overt

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FIG. 2. Correlation between predicted and observed rates of he-patic protein degradation during starvation and refeeding. Observedrates (e), expressed as. a percentage of fed control values, were takenfrom Fig. 1; predicted rates, similarly expressed, were computed fromthe absolute volumes of AVd + DBp (0) orAVd + totalDB (n) and mul-tiplied by (i) the average cytoplasmic protein concentration in hepa-tocytes (mean of lines C and D, Table 3) and (ii) the assumed first-orderturnoverconstant of internalized protein, 5.22 hr-' (5). Volumes of to-tal DB are given in Table 2; volumes of DBp for fed, 48-hr starved, and12- and 24-hr starved-refed mice were calculated to be 4.916, 2.319, 1.016,and 1.334 1.l per liver (34-g mouse), respectively (see text). The ob-served fed control rate (100%) was 11.4 mg hr-1/34-g mouse; corre-sponding predicted rates were 11.0 (mximum), 10.5 (average), and 10.0(minimum). The prediction shown by A is discussed in the text.

autophagy continued at postabsorptive levels in the absence offood intake is in agreement with observations of Pfeifer (13) andcan be attributed in large part to the maintenance of relativelynormal plasma amino acid concentrations (21). Its sharpsuppression with refeeding would be the expected result of anincrease in amino acid and insulin levels during intestinal ab-sorption (5, 21). But reasons for the decrease in microautophagyare not as apparent. Novikoff and Shin (4) have called attentionto the probable role of smooth ER in isolating small bits of cy-toplasm, and we have encountered type A lysosomes most fre-quently in areas filled with dilated smooth ER. Because thequantity of smooth ER per cell falls appreciably during star-vation (22), one might expect to see a progressive decline inmicroautophagic activity with starvation if in fact there is a con-nection between smooth ER and this process.

By 12 hr of refeeding, hepatocyte volume was fully restored,and large areas of cytoplasm contained newly synthesized gly-cogen. The most striking difference between starved-refed an-imals and the other groups was a sharp change in the distri-bution of type A particles. They were not found at the edge ofthe zones of glycogen as we typically see them, but instead werelocated almost exclusively in areas that had the general ap-pearance of cytoplasm in starved animals-i.e., glycogen wassparse and the smooth ER was vesiculated (Fig. 3). Internalizedglycogen was only occasionally noted in type A bodies, but sev-eral images contained ribosomes (Fig. 3).The apparent partition of microsequestrational activity dur-

ing cytoplasmic regain has two significant implications. First,if the only portions of cytoplasm susceptible of being inter-nalized were those that were carried over from starvation, itcould mean that we had overestimated predictions of proteindegradation during refeeding. When rates at 60 and 72 hr wererecalculated on the basis of the protein content in 48-hr starvedlivers (Fig. 2, ), the original discrepancy at 72 hr between ob-

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Proc. Natl. Acad. Sci. USA 80 (1983)

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FIG. 3. Early type A dense bodies in hepatocytes of 48-hr starved,12-hr refed mouse livers. (a) View at low magnification (x 10,000) oftwo type RDB (lines) and one early typeA DB (arrowhead), also shownin the Inset (x20,000). The granular material within the vacuole ap-pears to have been sequestered in situ. Residual clumps of glycogen, gl,which were intentionally unstained, show as clear areas; bc, bile can-aliculus. (b) Type A DB with vacuole containing ribosomes in a bed ofgranular material. The enclosing double membrane appears to havebeen formed from smooth ER and may be in the process of fusing (ar-rowhead). (x 38,000.) (c and d) Sections at two depths of an unusual DBprofile suggesting internalization of ribosomes and cytoplasmic matrixin situ. The flap-like membrane extension could represent smooth ER,an invagination of the lysosomal membrane (23), or both. Note the densematerial between the membranes at the tip of the extension. d also showsnumerous dilated tubules of smooth ER. (x30,600.)

served and calculated rates disappeared; little effect was notedat 60 hr, where agreement was already close. Second, it sug-gests that a large fraction of the total suppression of proteolysisduring cytoplasmic regrowth might be the consequence of growthitself. Resynthesis of RNA, glycogen, and ER occurs very earlyin refeeding (21, 22, 24), and for this reason alone, numerouspossibilities exist for a transient suppression of microauto-phagy. In view of the probable involvement of smooth ER inthe formation of type A particles during the postabsorptive pe-riod and starvation, the question of its role during refeeding isparticularly relevant. Unfortunately, because glycogen is closelyassociated with smooth ER (22, 25), its visualization is difficult

in refed states, in which the content of glycogen is high. How-ever, the characteristic branched tubular network can be ap-preciated, as we have observed, when overlying glycogen is re-moved during perfusion with glucagon. What the ultimateexplanation for the microautophagic suppression will eventuallybe is a matter for speculation at present, but available evidenceindicates that it could relate in some way to the restructuringof smooth ER or even to its physical relationship with glycogen.

The authors thank Ann Bryan, Chih-Ying Ho, -Carolyn Lloyd, DanMcBride, Kathleen Shiffer, and John Wert for their skillful technicalassistance; Dr. Bryce L. Munger for use of electron microscopic facilitiesin the Department of Anatomy; and Bonnie Merlino for typing themanuscript. This work was supported by U.S. Public Health Service GrantAM-21624 and a grant from the American Diabetes Association to N.J.H.

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