hplc analysis of insulin degradation products from isolated

HPLC Analysis of Insulin DegradationProducts From Isolated HepatocytesEffects of Inhibitors SuggestIntracellular and Extracellular PathwaysFREDERICK G. HAMEL, DANIEL E. PEAVY, MICHAEL P. RYAN, AND WILLIAM C. DUCKWORTH

SUMMARYIsolated rat hepatocytes were incubated withA14-[

125l]monoiodotyrosyl insulin for 30 min, and labeledmaterial was extracted from the cells and incubationmedia. The medium and the cell extract werechromatographed on a Sephadex G-50 column, andradioactivity eluting in the position of intact insulinwas concentrated and analyzed on HPLC. The HPLCanalysis of the cell extract showed two major productseluting from the column at 19 and 23 min, whereasmedium extracts showed one prominent producteluting at 14 min. Inclusion of chloroquine in theincubation blocked the formation of cellular productsat 19 and 23 min and caused the accumulation of aproduct eluting at 41 min while not affecting the mediaproducts. After sulfitolysis all cellular productscontained an intact A-chain. Dansylcadaverineincreased media products and altered the cell-extracted product pattern such that it had a major peakat 14 min, similar to media. These results suggest thattwo pathways for insulin degradation exist withinhepatocytes. The extracellular process forms productsthat are essentially unchanged by chloroquine anddansylcadaverine. The intracellular process is alteredby chloroquine and apparently inhibited bydansylcadaverine. Diabetes 36:702-708,1987

The mechanism(s) by which cells degrade insulin,the products that are formed during this process,and their site(s) of formation have not been estab-lished. Evidence has been presented that there are

two cellular pathways for insulin metabolism (1-4). One isan endocytotic process that results in the intracellular deg-

From the Veterans Administration Medical Center, Indianapolis, and the De-partments of Pharmacology and Toxicology, Medicine, and Physiology, In-diana University School of Medicine, Indianapolis, Indiana,Address correspondence and reprint requests to Frederick G. Hamel, PhD,1481 W. 10th Street (111E), Indianapolis, IN 46202.Received for publication 16 July 1986 and accepted in revised form 14 Jan-uary 1987.

radation of insulin (5,6); the other is a membrane processthat does not require intemalization (7,8). Although bothpathways apparently involve receptor binding of insulin asthe initial step (9), the subsequent steps, the enzymes in-volved, and the products formed by each are not clear.

After receptor binding, some of the insulin is internalizedthrough receptor-mediated endocytosis (10). The processgenerally involves clustering of receptor-bound ligand intocoated pits followed by invagination and pinching off of thepits from the plasma membranes. This results in the formationof cytoplasmic vesicles. The interior of these vesicles has arapid fall in pH due to proton pumps, which may result indissociation of receptor-bound ligand and facilitate pro-cessing and degradation of the ligand. Endocytotic vesiclesundergo sorting that may involve recycling to the membrane(diacytosis or retroendocytosis), interaction with other intra-cellular organelles (Golgi or nucleus), or delivery to lyso-somes for degradation. Although it is not fully established,insulin appears to be handled by this general process withsome cell-type to cell-type variations. A significant portionof receptor-bound insulin is internalized, with some of theinternalized insulin recycled to the membrane and releasedintact (diacytosis) (11). The remainder of the endocytosedinsulin is degraded. Although lysosomes may ultimately par-ticipate in degradation, it has been suggested that degra-dation may be initiated before insulin delivery to lysosomes,perhaps while the molecule is still within the endocytoticvesicle (12), as is true for other endocytosed ligands (13).It has been suggested that this process might play somerole in insulin action (14-16). One reason for the belief thatinsulin degradation may be initiated before lysosomes is thedemonstration of intracellular "insulin-sized" degradation in-termediate products (17,18). Although some studies suggestthat these intermediate products are formed on the mem-brane (7), we have recently presented evidence that theyare not accessible to the media (19). There is considerableevidence, however, that some degradation occurs on themembrane without requiring intemalization, but the mech-anism by which this might occur remains unclear.

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In this study we have approached these questions byexamining media and cellular insulin-sized products gen-erated by isolated hepatocytes. We used inhibitors of theendocytotic process to examine the effect of interruptingnormal processing on the formation of insulin degradationproducts.

MATERIALS AND METHODSDetails of our isolation and incubation procedures have beenpublished previously (17) and are briefly reviewed. Hepa-tocytes were isolated by a modification (14) of the methodof Terris and Steiner (9) with Krebs Improved Ringer II buffer.After isolation, ceils were first preincubated in buffer with 3%bovine serum albumin for 30 min before use. Cells were thenharvested by centrifugation and resuspended in fresh al-bumin-containing buffer. Labeled insulin and inhibitors were

' added either dissolved or diluted from a stock solution intothe incubation buffer. Cells were incubated for 30 min at37°C with A14-[

125l]iodoinsulin alone or with 200 |xM chloro-quine, 500 p-M dansylcadaverine, or a combination of chloro-quine and dansylcadaverine. Controls were also performedin which either a label or inhibitor was added after incubationto examine possible effects of extraction on the formation ofdegradation products. No such effects were seen. After in-cubation, the cell suspension was overlaid on a gradient ofsilicone oil (density 1.02) on an extraction mix consisting of7 M urea, 3 M acetic acid, 0.2% (vol/vol) Triton X-100, and100 ng/ml unlabeled pork insulin as carrier. After centrifu-gation for 1 min at 8700 x g, three phases resulted; cell-free buffer on top, silicone oil in the middle, and a lowerphase of cell extract and pelleted cell debris. An aliquot ofcell-free buffer was removed, and the tube was frozen inliquid nitrogen and severed just above the oil-extraction mixinterface. The tube tip and buffer samples had equal volumesof extraction mix added to them and were shaken for 18 hat 4°C. Extracted samples were centrifuged at 2000 x g for10 min at 4°C, and the supernatant was applied to a Seph-adex G-50 column (0.9 x 55 cm) and eluted with 1 M aceticacid. As reported previously (17), radioactivity eiuted fromthe Sephadex G-50 column in three peaks: a high-molecular-weight peak corresponding to the void volume, a peak inthe region corresponding to intact insulin, and a low-molec-ular-weight peak. Based on the elution profile, fractions con-taining insulin-sized material were pooled and lyophilized.

The lyophilized material was dissolved in 0.2 M ammoniumphosphate, pH 4.0, and 250 jxl was applied to a reversed-phase HPLC column. An aliquot was directly counted toestimate recoveries, and the relative degradation of the sam-ple was assessed by determination of its solubility in 10%trichloroacetic acid. The HPLC separations were performedwith a Beckman model 332 liquid chromatograph equippedwith a model 210 injector fitted with a 250-jxl loop. The col-umn used was a DuPont Zorbax C-8 (4.6 mm x 25 cm; 6-|xm particle diam) maintained at 40°C with a BioanalyticalSystems LC-22A temperature controller. Insulin and deg-radation products were eluted from the column with a 0.2-M ammonium phosphate (pH 4.0)/acetonitrile solvent sys-tem similar to that described previously (20). The isocraticand linear gradient steps in acetonitrile concentration usedwere: 7) 5 min at 23.75%, 2) 5-min gradient to 26.25%, 3)

TABLE iDistribution of radioactivity on Sephadex G-50 column fromisolated hepatocytes incubated with A14-['

25l]iodoinsulin

MediumControlChloroquineDansylcadaverineChloroquine plus

dansylcadaverineCells

ControlChloroquineDansylcadaverineChloroquine plus

dansylcadaverine

A

4.0 ± 0.34.1 ± 0.54.5 ± 0.4

4.1 ± 0.7

7.8 ± 1.48.8 ± 1.2

10.0 ± 2.4

6.6 ± 1.5

Peaks

B

83.2 ± 4.188.1 ± 3.781.8 ± 6.6

85.0 ± 5.2

81.0 ± 2.588.3 ± 2.881.4 ± 3.8

89.6 ± 2.3

C

12.9 ± 3.97.9 ± 3.1

13.7 ± 6.2

10.9 ± 4.7

10.3 ± 1.34.2 ± 1.57.5 ± 2.1

3.8 ± 0.8

Values expressed as percent recovered radioactivity (mean ± SE).Peak A is large molecular weight, peak B is insulin sized, and peakC is small molecular weight.

30 min at 26.25%, 4) 30-min gradient to 27.50%, 5) 15-mingradient to 40%, 6) 5 min at 40%, 7) 5-min gradient to23.75%, and 8) 5-min at 23.75%. Flow was maintained at1.0 ml/min throughout the elution. Fractions (0.5 ml) of theeluate were collected at 0.5-min intervals and counted di-rectly in a Tracor Analytic -y-counter to determine the elutionprofile of radioactivity.

Further analysis of degradation products was done withoxidative sulfitolysis and rechromatography. The radiola-beled products from the HPLC elution were collected andlyophilized. The dried material was dissolved in 1 M aceticacid, applied to a C-18 Sep-Pak, desalted with a water wash,and eluted with 3 ml of 80% acetonitrile/0.1% trifluoroaceticacid. The sample was lyophilized and dissolved in 0.2 Mammonium phosphate, pH 8.5, 7 M urea, and 2 mM EDTAwith 50 fxg pork insulin. Twenty milligrams each of sodiumsulfite and sodium tetrathionate were added, and the samplewas allowed to react at room temperature for at least 20 min.The sample was then injected onto the HPLC and run asdescribed previously (20).

Pork insulin, labeled with 125I at the A14 position (21), wasthe gift of Dr. B. Frank of Eli Lilly (Indianapolis, IN). Aceto-nitrile was HPLC grade purchased from Burdick and Jackson(Muskegon, Ml). The aqueous buffer was made with glass-distilled water, adjusted to pH 4.0, and filtered through a0.2-jxm Metricel membrane filter (Gelman Science, Ann Ar-bor, Ml). Chloroquine was purchased from either BoehringerMannheim (Indianapolis, IN) or Sigma (St. Louis, MO); dan-sylcadaverine was obtained from Sigma. All other chemicalswere of at least reagent grade.

RESULTSTable 1 shows the distribution of radioactivity on the Seph-adex G-50 column (Fig. 1) from control cells and controlmedia and the effect of chloroquine and dansylcadaverineseparately and together on the elution profiles. Resultsshown are means ± SE from four separate experiments.Chloroquine decreased degradation, as reflected by small-molecular-weight products in the medium (peak C), by anaverage of 40%. Dansylcadaverine had no effect on peak

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C products in the medium, but both inhibitors decreasedsmall-molecular-weight products intracellularly (P < .05 bypaired t test). No significant changes in the percentage ofhigh-molecular-weight materials were seen in the presenceof these inhibitors, but because chloroquine increases totalcell-associated radioactivity (by 75-200% in the 4 experi-ments), the absolute amount of radioactivity was increasedin the high-molecular-weight peak from chloroquine-treatedcells.

Figure 2 (top) shows the HPLC elution profile for insulin-sized material extracted from control cells. Most of the la-beled material eluted in the position of intact A14-[

125l]iodo-insulin, but a prominent and reproducible doublet can beseen at - 1 9 and 23 min as has been reported previously(17-19). In four separate experiments the percentage oftotal applied counts (mean ± SE) recovered was 3.85 ±0.37% in the 19-min peak and 3.86 ± 0.23% in the 23-minpeak, both of which agree with our previously publishedvalues (17). In addition, some material eluted after A14-[125l]iodoinsulin at ~85 min. Figure 2 {bottom) shows theelution pattern of insulin-sized material extracted from theincubation medium. Again, most of the labeled materialeluted in the A14-[

125l]iodoinsulin peak, but a prominent peak(7.39 ± 2.20% of total radioactivity) can be seen at 14 minand other smaller peaks elute both before and after A14-[125l]iodoinsulin. The pattern of elution of the medium prod-ucts is totally different from that of the cell, demonstratingthat extracellular and intracellular products are different.

Control experiments were done in which cells wereincubated without insulin for up to 2 h and centrifuged,and the conditioned medium was removed. The A14-[125ljiodoinsulin was then added and incubated for an ad-ditional 2 h. The medium was then chromatographed onSephadex G-50 column, and the insulin peak lyophilized andinjected on HPLC. As with media from cell incubation, a clearpeak could be seen at 14 min, but this peak comprised only0.5-0.7% of the total radioactivity (data not shown). Thus,conditioned medium contains a small amount of degradingactivity, but this activity, even with a much longer incubation,can explain only a small fraction (<10%) of the products

704

FIG. 1. Sephadex G-50 column elution profileof cell-bound radioactivity from isolatedhepatocytes treated with chloroquine. First elutedpeak (fractions 13-14) is high-molecular-weightmaterial; second eluted peak (fractions 21-28) isinsulin-sized material; third eluted peak (fractions39-45) is low-molecular-weight material.Distribution of radioactivity in the 3 peaks isshown in Table 1 for cells and media for allexperimental conditions.

obtained from the cell incubation. These results suggest thatmost of the media products are indeed being generated byinsulin interacting with the cells.

Preliminary studies were done to examine the time courseof the generation of the major insulin-sized products fromcells and media. From extracted cells an appreciable prod-uct was not present after 2 min of incubation at 37°C, butby 5 min the doublet was present with 3.48% of the countseluting at 19 min and 3.57% at 23 min. The proportions ofcellular counts eluting as this doublet remained essentiallyconstant for 60 min of incubation. In these experiments, ap-

2.0 -

- . '-6 -

y ,240-0- O.8-<

O 0.4-

wv L

0 20 40 60 80 I00

ELUTION TIME (MIN)

FIG. 2. HPLC of insulin-sized radiolabeled material isolated fromcells (top) and media (bottom) of hepatocytes incubated withA14-[

12Sl]iodoinsulin.

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o.o20 40

ELUTION6 0

TIME

80

(MIN)

100

FIG. 3. HPLC of insulin-sized radiolabeled material isolated fromcells (top) and media (bottom) of hepatocytes incubated withA14-[

125l]iodoinsulin and 200 JAM chloroquine.

preciable insulin-sized products from the medium were notseen until 15 min, and the amount of material that eluted inthe 14-min peak increased from 1.53% at 15 min to 6.83%by 60 min. Total media degradation, as reflected by small-molecular-weight products on the Sephadex G-50, was alsofirst seen at 15 min and increased to 30.2% of the total countsby 60 min.

For further examination of the time course of product gen-eration, cells were incubated with A14-[

125l]iodoinsulin at 15°Cfor 4 h. At 15°C the internalization of insulin is markedlydecreased, and most of the bound hormone remains at thecell surface (22). Under this condition, by 1 h the intracellulardoublet was present but in reduced amounts compared withthose at 37°C. Percentage of total counts eluting at 19 minwas 1.43% and at 23 min was 1.81%. These percentagesremained constant for up to 4 h. In the medium from 15°Ccells, a very small peak eluting at 14 min was seen at 1 h,comprising only 0.65% of total counts. Over 4 h this fractionincreased to 1.95% of total counts. By measurement assmall-molecular-weight material on the Sephadex G-50, totaldegradation at 4 h was 10%.

Incubation of A14-[125l]iodoinsulin with hepatocytes in the

presence of chloroquine followed by cell extraction andHPLC separation of insulin-sized products resulted in thepattern shown in Fig. 3 (top). The prominent doublet seenwith control cells is totally absent. Two new peaks, not pres-ent in control cells, can be seen at 27 and 41 min. The 27-min peak comprised 0.81 ± 0.11% (mean ± SE of 4 ex-periments) of the total radioactivity. The 41-min peak wasfound in three of the four experiments but was not found inthe one experiment in which degradation, as reflected bythe generation of low-molecular-weight material, was moreextensive. In the three experiments in which this peak was

found, it represented 2.71 ± 0.26% of total counts. Chlor-oquine has been reported to block the processing of en-docytosed insulin, resulting in the accumulation of cyto-plasmic vesicles containing insulin (23). The pattern shownin Fig. 3 (top) suggests that chloroquine blocks the formationof the less hydrophobic products eluting as the doublet at19 and 23 min while causing the accumulation of productsthat elute from HPLC at 27 and 41 min. Cells in which moreextensive degradation has occurred in spite of chloroquinedo not contain the 41-min product. The HPLC pattern of theinsulin-sized material taken from the incubation medium ofchloroquine-treated cells is shown in Fig. 3 (bottom). Chlor-oquine produces no change in this pattern from the controlpattern, supporting the concept that the medium material isnot derived from an intracellular pathway.

To further examine the question of size and compositionof these products, additional experiments were done. Chlor-oquine-treated cells were incubated with A14-[

125l]iodoinsulinand extracted and chromatographed as usual. Instead ofpooling the Sephadex G-50 insulin peak, however, individualfractions from different areas of this peak were injected onHPLC and the elution examined. Table 2 shows the resultsof this experiment. The Sephadex G-50 column elution of the41-min peak corresponds to the elution of intact insulin, sug-gesting it is the same or very similar in size to intact insulin.The 27-min peak, however, is shifted to later fractions com-pared with intact insulin, suggesting it is somewhat smaller.

The next experiment was done to further examine the na-ture of the products in control and chloroquine-treated cells.Cells were extracted and chromatographed on the Sepha-dex G-50 and then injected on HPLC. The three peaks fromchloroquine-treated cells (27 min, 41 min, and intact A14-[125l]iodoinsulin at 55 min) were pooled, lyophilized, and sul-fitolized. The sulfitolized material was reinjected on HPLCwith a program for isolating the A-chain. The radioactivematerial from all three peaks eluted in the position of intact,sulfitolized A-chain (Fig. 4). The products from control cells(19- and 23-min peaks and intact insulin) were also sulfito-lized and reinjected on HPLC. These products also elutedin the position of authentic A-chain (Fig. 4), showing that inboth control and chloroquine-treated cells the major cellularproducts contain intact A-chain. These results demonstratethat the metabolism of insulin by hepatocytes occurs withinitial alterations in the B-chain (24,25).

Dansylcadaverine blocks cellular processing of insulin ata point before the chloroquine-sensitive step (14). The HPLC

TABLE 2Distribution of radioactivity on HPLC of individual Sephadex G-50column fractions obtained from chloroquine-treated hepatocytes

Sephadexfraction

1819202123242526

Insulin peak(55 min)

236111,30119,69731,19916,73810,27350693139

27-min peak

000

109221345325168

41-min peak

026548710644032589235

Values are counts per minute.


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80 -i

70 "

60 "

50 "

ZZ 30 -

<oQ<DC

20 -

10 "

10 20 30 40 50

ELUTION TIME (min)

FIG. 4. HPLC of sulfitolyzed A14-[125l]iodoinsulin recovered from

isolated hepatocytes. Identical elution patterns were obtained with27- and 41-min peaks from chloroquine-treated cells, 19- and23-min peaks from control cells, and A14-[

125l]iodoinsulin standardthat was not exposed to cells or media.

pattern of insulin-sized material extracted from dansylca-daverine-treated hepatocytes is shown in Fig. 5 (top). Boththe 19- and 23-min doublet seen in control cells and the 41-and 27-min peaks formed in the presence of chloroquineare missing. Instead, a peak eluting at 14 min is seen. Thispattern is similar to the media pattern seen both with thecontrol (Fig. 2, bottom) and the dansylcadaverine-treated(Fig. 5, bottom) cells. In dansylcadaverine-treated cells, themedium contains more of the intermediate product at 30 min(10.14 ± 2.9 vs. 5.68 ± 3.0% in control cells), suggestingthat cellular degradation is altered in the presence of dan-sylcadaverine so that more of the insulin is shunted throughthe membrane-medium pathway. In the presence of bothchloroquine and dansylcadaverine, the pattern of productswas identical to that with dansylcadaverine alone, furthersupporting the concept that these two inhibitors have effectson the same pathway with dansylcadaverine working at asite proximal to that of chloroquine.

DISCUSSIONThese results demonstrate that insulin-sized degradationproducts are present both in the medium and intraceilularlyin isolated hepatocytes and that products found in the me-dium are different from those found in the cell. The majorcellular intermediates consist of a pair of products that elutefrom our HPLC system as a doublet at 19 and 23 min, withintact A14-[

125l]iodoinsulin eluting at 55 min. Although the size

of these products is approximately that of insulin, based onSephadex G-50 column elution, they are markedly less hy-drophobic than the intact molecule. This suggests either amajor change in surface charge or a loss of hydrophobicresidues. Although conformational changes will result in analteration in elution from a reversed-phase column, it seemsunlikely, given the hydrophobic core of the insulin molecule,that these products result simply from conformationalchanges. Sulfitolysis of the labeled products demonstratesthat the A-chain is intact, suggesting that the B-chain is thesite of the initial degradative step. This is consistent withseveral previous reports of insulin degradation products withloss of a portion of the B-chain (24,25). These products areformed within 5 min of exposure of A14-[

125l]iodoinsulin tohepatocytes at 37°C and are present through at least 2 h ofincubation in relatively constant amounts. These productsare also present at 15°C, at which temperature internalizationis markedly reduced (22), further supporting the hypothesisthat these are prelysosomal (26,27).

The formation of these products is totally inhibited by chlor-oquine. Although chloroquine may block several steps inthe endocytotic pathway, including lysosomal degradation,there is persuasive evidence that the major effect of thisagent is on the initial endocytotic (clear, unique) vesicle (23).Chloroquine prevents acidification of the endosome, re-sulting in accumulation of intracellular vesicles contain-ing insulin. This results in inhibition of intracellular insulindegradation as reflected by the formation of small-molecular-weight products and, as shown, prevention of the formationof the characteristic intracellular insulin-sized products. Ourresults show, however, that chloroquine does not totallyblock degradative steps. Instead, different insulin-sized

2 . 0 -

QUJ_ lQ_Q_<

S?

1 . 6 -

.2 -

0.8-

0.4-

0 .0

2.0 -

.— I

0 . 8 -

.2 -

0 . 4 -

0-00 20 40 60

ELUTION TIME (MIN)

FIG. 5. HPLC of insulin-sized radiolabeled material isolated fromcells (top) and media {bottom) of hepatocytes incubated withA14-[

125l]iodoinsulin and 500 fiM dansylcadaverine.

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products are formed. These products are less hydrophobicthan intact insulin but more hydrophobic than the primaryproducts found in control cells. They also have an intact A-chain, suggesting an alteration in the B-chain. The morehydrophobic of the two products coelutes with insulin fromthe Sephadex G-50 column, whereas the 27-min productelutes slightly later, suggesting that a fragment of the B-chainhas been lost. These results suggest that in the presence ofchloroquine different insulin degradation intermediates areproduced or that chloroquine allows the accumulation ofdegradation intermediates that normally are present onlytransiently and in small amounts.

Dansylcadaverine also blocks receptor-mediated endo-cytosis (10). Although the mechanism of its action is evenless clear than that of chloroquine, it apparently blocks at astep proximal to that of chloroquine, i.e., before the formationof the endocytotic vesicle (14). One suggestion has beenthat it allows invagination of the coated pit but prevents thepinching off from the membrane to form the cytoplasmicvesicle. The pit may or may not maintain a functional ex-posure to the outside of the cell. If it does not, then duringcentrifugation for separation of cells from the medium, thecontents of the pit would segregate with the cell, and thus,products that would normally be released into the mediawould remain apparently cell associated. This appears tohappen. In the presence of dansylcadaverine, none of thetypical cell-associated insulin-degradation products can befound. Neither the products seen in control cells nor theproducts found in chloroquine-treated cells are present. In-stead, the product pattern found in the medium is seen, witha major 14-min peak on HPLC. Another possibility is that themedia products are increased in the cell pellet due to non-specific association; however, this is highly unlikely becausedansylcadaverine did not alter the inulin space of hepato-cytes (unpublished observation).

Medium products are different from cell-associated prod-ucts. The major product elutes from HPLC at 14 min, makingit even less hydrophobic than cellular products. Chloroquinehas no effect on this, but dansylcadaverine produces a sig-nificant increase in this material. This supports the conceptthat dansylcadaverine allows continued exposure of recep-tor-associated ligand to an external environment functionallyshunting the degradative pathway away from intracellularprocessing, allowing more membrane degradation to occur.In this regard an important question remains: Does the ex-ternal degradative system involve membrane interaction oris it due to extracellular proteolytic processes? The mediumfrom incubated cells contains variable amounts of insulin-degrading activity (1). In many isolated cell preparations,most insulin degradation, as reflected by the formation ofsmall-molecular-weight products, occurs as non-cell-asso-ciated degrading activity. Bacitracin has been used to inhibitthis extracellular degrading activity, but we and others haveshown that bacitracin can also affect cellular degradation(8,17). Cultured cells can also be used because the mediumfrom cultured cells has little or no detectable (by TCA assay)degrading activity (28). With careful attention to cell prep-aration, freshly isolated hepatocytes can be obtained thatdo not "leak" apparent degrading activity into the medium.Medium taken after exposure to the cells used in our studydoes not produce TCA-soluble materials from A14-[

125l]iodo-

insulin after an additional 2-h incubation. This medium, how-ever, does produce a small amount of insulin-sized inter-mediate products. This suggests that a small amount ofdegrading activity is "shed" from our incubated cells. Theamount of medium activity can explain only a small portionof the medium product. The presence of cells is required toobtain the quantity of product typically seen, suggesting thatmost of the medium insulin-sized intermediate products re-quire interaction of insulin with the cell. Several studies haveshown the presence of insulin-degrading activity on the ex-ternal surface of the cell membrane (29). Thus, this exofacialdegrading activity may be involved in the production of me-dium products. Some of this activity may also be shed intothe incubation medium. We can detect this activity even inthe medium of cultured hepatocytes, both adult and fetal,and can produce alterations in amounts, both increased anddecreased, by using various inhibitors (unpublished obser-vations). This may suggest that shedding of degradativeactivity occurs regularly and possibly could serve somephysiological function. Regardless, most of the insulin-sizedmedium products in our studies appear to derive from cell-associated processes.

Other studies with these inhibitors have found alterationsin cellular insulin degradation. Kitabchi and Stentz (16) re-cently examined the effect of inhibitors of insulin degradationon intracellular products in human fibroblasts. In contrast toour studies, they did not find a decrease in intermediateproducts in the presence of chloroquine or dansylcadaverinebut did find a decrease in the amount of iodotyrosine formedin the presence of these inhibitors. The reason for this dif-ference is not immediately apparent but may be related tothe cell type, because fibroblasts and hepatocytes may wellhandle insulin differently.

Our results are compatible with two degradative pathwaysfor insulin in isolated hepatocytes, which produce differentinsulin-sized intermediate products. Inhibitors of cellulardegradation do not affect the pattern of media products,suggesting that the two pathways are distinct. The relativeroles of the two pathways, the enzymes involved, and theirphysiological importance are uncertain. Inhibition by dan-sylcadaverine of some, but not all, of insulin's actions sug-gests that the intracellular pathway may be in some wayinvolved in some of insulin's actions. The existence of twopathways may also explain some of the apparently discor-dant results among various studies as to the effect of inhib-itors on cellular insulin degradation.

ACKNOWLEDGMENTSWe thank Dr. Juris Liepnieks for valuable discussions andtechnical assistance and Jeanne Braunwarth for technicalassistance.

This work was supported in part by a Veterans Adminis-tration Merit Review (W.C.D.) and a Diabetes Research andTraining Center Pilot Project (F.G.H.).

Parts of this work were presented at the annual meetingsof the American Diabetes Association in 1984 in Las Vegas,NV, and in 1985 in Baltimore, MD.

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2. Draznin B, Solomons CC, Toothaker DR, Sussman KE: Energy-dependentsteps in insulin-hepatocyte interaction. Endocrinology 108:8-17, 1981

3. Draznin B, Todd WW, Leitner JW, Toothaker DR: Lysosomal and non-lysosomal pathways of intracellular insulin degradation in isolated rathepatocytes. Horm Res 15:252-62, 1981

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5. Gliemann J, Sonne 0: Binding and receptor mediated degradation ofinsulin in adipocytes. J Biol Chem 253:7857-63, 1978

6. Gorden P, Carpentier JL, Freychet P, LeCam A, Orci L: Intracellular trans-location of iodine 125 labeled insulin: direct demonstration in isolatedhepatocytes. Science 200:782-84, 1978

7. Blackard WG, Ludeman C, Stillman J: Role of hepatocyte plasma mem-brane in insulin degradation. Am J Physiol 248:E194-202, 1985

8. Hammons GT, Smith RM, Jarett L: Inhibition by bacitracin of rat plasmamembrane degradation of 125I insulin is associated with an increase inplasma membrane bound insulin and a potentiation of glucose oxidationby adipocytes. J Biol Chem 257:11563-70, 1982

9. Terris S, Steiner DF: Binding and degradation of 125I insulin by rat he-patocytes. J Biol Chem 250:8389-98, 1973

10. Wileman T, Harding C, Stahl P. Receptor mediated endocytosis. Biochem./232:1-14, 1985

11. Marshall S: Dual pathways for the intracellular processing of insulin. Re-lationship between retroendocytosis of intact hormone and the recyclingof insulin receptors. J Biol Chem 260:13524-31, 1985

12. Juul SM, Jones RH, Evans JL, Neffe J, Sonksen PH, Brandenburg: Evi-dence for an early degradative event to the insulin molecule followingbinding to hepatocyte receptors. Biochim Biophys Acta 856:310-19,1986

13. Diment S, Stahl P: Macrophage endosomes contain proteases whichdegrade endocytosed protein ligands. J Biol Chem 260:15311-17, 1985

14. Peavy DE, Edmondson JW, Duckworth WC: Selective effects of inhibitorsof hofmone processing on insulin action in isolated hepatocytes. Endo-crinology 114:753-60, 1984

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hplc analysis of insulin degradation products from isolated

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