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The effects of low temperature and chloroquine on 125I-insulin degradation by the perfused rat liver
ARCHIVES OF BIOCHEMISTRY Vol. 212, No. 1, November,
The
Effects
of Low Temperature and Chloroquine on ‘251-lnsulin Degradation by the Perfused Rat Liver’
PATRICIA Prmm
AND BIOPHYSICS pp. 1’70-176, 1981
A. DENNIS
NATHAN
AND
N. ARONSON,
in Biodwmistry, Althouse Laboratory, The Penns@ania Unhrsit~ Park, Pennsylvania 16802 Received
June
State
JR. University,
28, 1981
Low temperature and the lysosomotropic agent, chloroquine, were used to study the degradation of ?-insulin in a perfused rat liver. Insulin (1.5 X 10-$&i) was removed from the perfusate at 35°C with a Tllz of 12 min, and this process was slowed to 35 min at a temperature of 17°C. Essentially no degradation of ‘=I-insulin took place in the liver at 17°C. After 90 min at that temperature 64% of the liver radioactivity had accumulated in the microsomal fraction of the tissue homogenate, while at 35°C 60% of the radioactive material was in the supernatant fraction. Greater than 80% of the supernatant radioactivity was acid soluble. Rapid warming of a 17°C-treated liver to 35°C allowed the accumulated ‘=I-insulin in the microsomal fraction to be degraded to acid-soluble products in the normal manner. Chloroquine (0.2 mM) also caused the liver to degrade insulin more slowly. At 60 min after adding lzI-insulin to the chloroquine-treated liver, 50% of the radioactivity in the tissue was still present in the lysosome-rich fraction of the homogenate, while less than 10% was in this fraction in a control liver. The effects of low temperature show transfer of insulin to its degradative site is rate limiting for hormone catabolism and the inhibition by chloroquine suggests lysosomes have a role in insulin degradation by the liver.
A major site of insulin degradation in the body is the liver (1, 2). The hormone is initially bound by specific receptors on the cell surface, then becomes internalized and subsequently digested. Although these events have been demonstrated in the liver in viva (3, 4), in the perfused organ (5, 6), and in isolated hepatocytes (7, 8), as well as in other cell types (g-12), there is uncertainty regarding the exact intracellular location(s) of insulin breakdown. Insulindegrading systems have been found in the microsomal (13), cytoplasmic (14,15), and plasma membrane fractions (16) of liver homogenates, and lysosomal hydrolases obtained from this tissue are capable of rapidly digesting the peptide hormone in vitro (17, 18). Biochemical and electron 1 This work was supported by Grant from the National Institutes of Arthritis, lism, and Digestive Diseases of the United lic Health Service. 0003-9861/81/130170-07$02.00/o Copyright (B 1981 by Academic All rights
of reproduction
Preu. Inc. in any form reserved.
AM-15465 MetaboState Pub170
microscopic evidence have suggested several possible routes for the internal processing and catabolism of insulin: (a) a degradative pathway via lysosomes that is implicated in both hepatocytes and adipocytes (3, 19, 20); (b) an initial interaction with the Golgi apparatus as demonstrated in the liver (4, 21, 22); and (c) an association of the hormone with the nucleus and endoplasmic reticulum of cultured lymphocytes (11). In this current work we have studied the effects of lysosomotropic agents and low temperature on the metabolism of ‘%I-insulin by the perfused rat liver in order to substantiate that lysosomes are important for physiological degradation of this hormone. Lysosomotropic agents are compounds which accumulate in lysosomes and disrupt their functioning either by inhibiting specific hydrolytic enzymes or by changing the intralysosomal environment. These compounds have been reported to
EFFECTS OF TEMPERATURE
AND CHLGRGQUINE
partially decrease the breakdown of insulin in hepatocytes (23), adipocytes (20, 24,25), and fibroblasts (26). Mortimore and Tietze (5) several years ago reported that the normal degradation of ‘Y-insulin by a perfused rat liver could be prevented by maintaining the tissue at a cold temperature (5°C). Possibly relevant to their observation, our laboratory has recently shown that a perfused liver at 20°C stopped degrading endocytosed ‘2SI-asialofetuin (27). This glycoprotein enters hepatic lysosomes after its receptor-mediated uptake by the liver, and inhibition of its degradation at 20°C was caused by the failure of pinocytic vesicles carrying the glycoprotein to fuse with the lysosomes. Insulin that becomes internalized after binding to its receptor may also be prevented from entering the lysosomes by a similar inhibition at some critical low temperature. The experiments reported here using lysosomotropic substances and low temperature indicate that lysosomes do participate in insulin degradation by the liver, but other intracellular routes for catabolism of this important pancreatic hormone cannot be excluded. EXPERIMENTAL
PROCEDURES
Preparation of ‘25I-labeled insulin Porcine insulin (Sigma Chemical Co., 24 units/mg) was iodinated by a modification of the chloramine T method of Terris and Steiner (8). The reaction was done at room temperature in 0.1 ml of 0.3 M sodium phosphate buffer, pH 7.0. Insulin (2.0-5.0 nmol) and 1.5 mCi of carrierfree Na? (1’7 Ci/mg, New England Nuclear Corp.) were reacted upon addition of chloramine T (Eastman Kodak) equivalent to the nanomoles of insulin. After 3 to 3.5 min, 2 nmol of sodium metabisulfite/ nmol of chloramine T was added to stop the reaction, which was then mixed with 0.3 ml of 4% bovine serum albumin and run through a column (1.5 x 5.0 cm) of Sephadex G-25. The column was preequilibrated and eluted with 0.01 M Tris-HCl buffer, pH 7.0, that contained 0.05~ NaCl and 0.25% bovine serum albumin. The eluted ‘WI-insulin was placed in dialysis tubing (Spectrum Medical Industries, Inc.) and dialyzed overnight in the cold against three changes of 0.1 M sodium phosphate buffer, pH 7.0. From 95 to 98% of the radioactivity in the iodinated protein (20-70 &i/pg) both precipitated with acid (see below) and comigrated with an insulin standard on polyacrylamide gels run in sodium dodecyl sulfate
ON INSULIN
DEGRADATION
171
buffer (23). Unlabeled insulin was used to dilute this product to a final specific radioactivity of 3 to 4 &i/m. !Fre&wnt of livera Rat livers were perfused cyclically at various temperatures as previously described (27) except 2% bovine serum albumin (Fraction V, Miles Laboratory) replaced polyvinylpyrrolidone in the perfusion medium (29). Fractionation of liver homogenates, including the isolation of purified lysosomes from Tribn-treated animals (30), was according to the procedures of de Duve and his colleagues (31). The molecular weight properties of the radioactivity within subcellular fractions was analyzed on a Sephadex G-50 column (1 X 50 cm) according to the method of Terris and Steiner (8). In vitro &&w&s qf ‘ysI-insulin Digestion of ‘%Iinsulin by purified lysosomal extracts (29) was done at 37°C in 0.6 ml of 0.1 M sodium acetate buffer, pH 5.0, containing 1.0 mlb reduced glutathione. In each experiment 1 H of insulin substrate was treated with 0.155 mg of protein from the lysosomal extract. At timed intervals 20-~1 samples of the reaction were diluted with 180 pl of 0.1 116phosphate buffer, pH 7.0, that contained 1% bovine serum albumin, and 1.0 ml of cold 4% phosphotungstic acid in 2 N HCl was added to precipitate the remaining ‘l-insulin. The precipitate was centrifuged and washed once with 1.0 ml of the acid solution. The radioactivity in separate samples of the combined supernatants and the redissolved precipitate (1.0 ml of 1.0 NNaOH) was measured in a Beckman Gamma 7030 spectrometer. Analytical procedures. Protein was assayed according to the method of Miller (32) using bovine serum albumin as a standard. Chloroquine and various substrates for enzyme assays were obtained from Sigma Chemical Company. Leupeptin was purchased from Protein Research Foundation (Minohshi, Osaka, Japan). RESULTS
Effects of temperature on rndabolh of 1e51-insdin by the perfused liver. The me-
tabolism of ‘%I-insulin by the perfused rat liver was examined over the temperature range from 5 to 35°C. At 35°C the removal of ‘%I-insulin (1.0 Fg) from the per&sate occurred with a Tl12 of 12 min (Fig. 1). Acid-soluble products began to appear in the perfusate 5 to 10 min after adding the protein, and by 1 h of perfusion almost complete release of liver radioactivity was attained. Liver uptake of lSI-insulin slowed progressively at lower temperatures; for example, the Tlj2 for removal was 35 min at 17°C. A more striking effect on insulin
172
DENNIS AND ARONSON
FIG. 1. Uptake and degradation of ‘?-insulin by the perfused liver at 35°C. Livers were perfused at 35’C for 30 min after addition of 1.0 1(8 of ‘?-insulin. Samples of perfusate (0.2 ml) were removed at various times and subjected to acid precipitation as described under Experimental Procedures. Acid-precipitable radioactivity (0); acid-soluble radioactivity (0). The acid-precipitable data were corrected for the amount (25-3056) of precipitable radioactivity which was never cleared from the perfusate. This acid-precipitable material may represent the ‘“I-insulin not recognized by the liver due to damage from the iodination procedure (5).
degradation occurred over this range of temperature (Fig. 2). Only 10% destruction of the added lSI-insulin took place after 90 min of perfusion at 17°C. This
small amount of acid-soluble products appears to be the minimum level of hormone breakdown that occurs in this experimental system for the same value was obtained at 5°C. In order to determine which cellular compartments were involved in the processing of ‘%I-insulin, radioactivity was measured in subcellular fractions isolated from liver homogenates that had been prepared after perfusing the tissue for 90 min at various temperatures. Most of the radioactivity was found in the microsomal fraction at 17’C (64% of the liver radioactivity), while at higher temperature the radioactive material in the liver shifted into the soluble fraction of the homogenate (60% at 35”C, Fig. 3). A parallel release of radioactivity from both the liver itself and the microsomal fraction occurred over this experimental range of temperature, while only a small amount of the homogenate radioactivity was ever located in the nuclear (7-13%), mitochondrial (2-6%), or lysosomal (3-10s) fraction. Preliminary examination of autoradiographs obtained from a liver that had been perfused 90 min at 17°C with ‘2SI-insulin has shown that at least half of the silver grains that formed were located intracellularly. The remainder of the silver grains were still present along hepatocyte surfaces. Thus, the accumulated microsomal radioactivity at 17°C apparently
70r------l
FIG. 2. Effect of temperature on the degradation of ‘l-insulin by the perfused liver. Livers were equilibrated at the indicated temperatures and then exposed to 1.0 pg of ‘“I-insulin for 30 min. At that time 500 pg of native insulin were added for 5 min to release any labeled insulin still bound to surface receptors. The livers were then perfused briefly with cold 0.25~ sucrose, homogenized, and subjected to subcellular fractionation. The combined acid-soluble radioactivity in the perfusate and liver homogenate was measured (0) and used to detemine overall degradation of the added insulin.
FIG. 3. Effect of temperature on subcellular distribution of radioactivity in the perfused liver. Livers were treated as described in Fig. 2. Total radioactivity in both the homogenate and each of its subcellular fractions was measured: microsomal fraction, (0); final supernatant, (0).
EFFECTS
OF TEMPERATURE
AND CHLOROQUINE
FIG. 4. Reversal of the cold temperature effect on ?-insulin metabolism by the perfused liver. A liver was equilibrated at 17°C and perfused with 0.1 gg of ‘“I-insulin for 30 min. At that time (light arrow) 500 c(g of native insulin was added for 30 min to remove surface bound ‘%I-insulin. At 130 min (heavy arrow) the liver was rapidly warmed to 35°C by switching to a new, prewarmed perfusion medium. Samples of perfusate (0.2 ml) were removed at the indicated times and their content of either acid-precipitable radioactivity at 1’7’C (0) or acid-soluble radioactivity at 35°C (0) was measured.
represents both internalized and surfacebound ‘251-insulin. The latter material is not easily exchanged by large amounts of unlabeled insulin (see Fig. 4). Our laboratory previously had shown that rewarming a cold perfused liver to 35°C reestablished the normal degradation of endocytosed asialofetuin (27). Such a reversal experiment was done using ‘%Iinsulin (Fig. 4). The liver took up greater than 65% of a O.l-pg sample of radioactive insulin after 90 min of perfusion at 17°C. Adding 500 .gg of native insulin at that time caused the organ to release 28% of its previously accumulated radioactivity. Rapid equilibration of the liver to 35°C allowed normal degradation of this accumulated material. Greater than 80% of the radioactivity released into the perfusate after warm-up to 35°C was acid soluble. Effect of lysosomotropic compwtmd.s on x251-insulin degradation Leupeptin, a thiol proteinase inhibitor, has been shown to be very effective in slowing the hepatic degradation of endocytosed asialofetuin (29). However, this peptide did not alter the
ON INSULIN
DEGRADATION
173
uptake and digestion of 1.0 pg ‘ZI-insulin by the liver during 90 min of perfusion at 35°C (data not shown). Inhibition was also not observed when reactions were done in vitro using 10T3 M leupeptin and purified lysosomal extracts (Fig. 5). However, Fig. 5 does show that lysosomal breakdown of insulin to acid-soluble products was pH dependent. Hydrolysis was much reduced at pH 6.8 compared to pH 5.0. On the basis of this result we tested the effects of chloroquine and ammonium ion on insulin metabolism by the perfused liver, since these substances have been shown by others to increase lysosomal pH (34, 35) and to inhibit insulin catabolism in adipocytes (20, 24, 25), hepatocytes (23), and fibroblasts (26). Chloroquine slowed the degradation of insulin by the liver (Table I). Compared with control livers this compound caused a threefold greater amount of radioactivity to be left in the tissue 60 min after adding ‘?-insulin to the perfusate, and twice as much of the accumulated radioactivity could be precipitated with acid. NH,+ caused similar, but somewhat reduced, changes (data not shown). The subcellular distribution of radioactivity was determined for a chlo-
FIG. 5. Effects of leupeptin and pH on the digestion of ‘l-insulin by lysosomal extracts in vitro. Incubations were done as decribed under Experimental Procedures under varying experimental conditions. Aliquots were removed at the indicated times and immediately subjected to acid precipitation. The amount of acid-soluble radioactive products formed is expressed as a percentage of the total acid-precipitable radioactivity present in the ‘l-insulin substrate at 0 min: control digest at pH 5.0, (0); pH 6.0, (m); pH 6.8, (A); lo-’ M leupeptin at pH 5.0, (0).
174
DENNIS AND
ARONSON
TABLE
I
EFFECT OF CHLOROQUINE ON 12SI-I~~~~~ METAEWLISM IN THE PERFUSED LIVERY Radioactivity
in the liver
Subcellular
Treatment Control (‘“I-insulin Chloroquine
of liver only)
Percentage of injected
Percentage acid soluble
8.0 21.4
41.8 17.4
distribution
(o/o of homogenate)
Nuclear
Heavy and light mitocbondrial
Microsomal
13.2 11.8
7.0 48.8
36.0 20.5
Supernatant 44.1 18.7
’ Livers were pretreated with 0.2 mM chloroquine for 60 min prior to addition of 1.0 pg of ‘“I-insulin. After a further 60 min of perfusion the livers were homogenized and subjected to subcellular fractionation. Acid precipitation of samples (0.2 ml) of each liver homogenate was done as described under Experimental Procedures. The data shown are the average values obtained from two experiments.
roquine-treated liver after 60 min of perfusion with ‘%I-insulin (Table I). Chloroquine caused a buildup of radioactivity within the lysosome-rich fraction, ML. The chloroquine-treated liver contained 21% of the injected insulin radioactivity at that time, while the control liver contained only 8%. Neither chloroquine nor NH,+ affected the rate of uptake of *=Iinsulin by the perfused liver. DISCUSSION
Our laboratory has recently shown that temperature can be useful for studying the process of receptor-mediated degradation of asialoglycoproteins by the perfused liver (27). Catabolism ,of ‘251-asialofetuin was found to cease below 20°C and this effect was caused by a specific inhibition of the fusion of endocytic vesicles with lysosomes. The temperature studies we have now done in the perfused liver with ?insulin show this peptide hormone to have a similar, but not exact, behavior as asialofetuin. Insulin degradation was maximal between 30 and 35”C, but diminished rapidly from 27 to 20°C (Fig. 2). As already shown for asialofetuin, 1251-insulin accumulated within the microsomes at the lower temperatures (Fig. 3). In a liver perfused at 17”C, 94% of this microsomal radioactivity was acid pxecipitable and greater than 33% eluted from gel filtration columns the same as intact insulin.
Almost 50% of this insulin appears assoicated with plasma membrane even though a large amount of unlabeled insulin had been added to the perfusate prior to homogenization (unpublished autoradiographic data). The remaining radioactive material was intracellular and may be associated with pinocytic vesicles, or with other subcellular membrane components (Golgi, endoplasmic reticulum). Ward and Mortimore (36) have recently reported the isolation of vesicles containing intact insulin from a liver perfused at 22°C. These vesicles had a similar density to lysosomes, but unlike the lysosomes, did not accumulate injected Triton WR-1339. Possibly these insulin containing structures are enlarged pinocyt-ic vesicles. The nearly total inhibition of insulin breakdown observed at 17°C could be reversed by warming the liver to 35°C (Fig. 4). As the liver temperature was raised toward physiological the radioactivity shifted from its microsomal location to the supernatant fraction of the homogenate (Fig. 3). The radioactive material in the sol.uble fraction was found to be mainly degradation products with greater than 90% eluting in the small-molecular-weight fraction from a Sephadex G-50column. No significant intermediate accumulation of radioactivity was seen in any other subeellular fraction. Thus, hydrolysis of insulin within the actual degradative com-
EFFECTS
OF TEMPERATURE
AND
CHLOROQUINE
ON INSULIN
DEGRADATION
175
partment and release of its products into pathway in hepatocytes. Recent evidence the cytoplasm were both rapid steps relalso suggests that lysosomotropic agents ative to transfer of the hormone from the (chloroquine, ammonium ion) can disrupt cell surface or vesicles into the catabolic other subcellular events, such as memsite. Whatever cellular structures are in- brane recycling (38) and fusion of vesicles volved directly in insulin breakdown, the (39, 49). It is, therefore, important to inrate-limiting step for this metabolism oc- terpret results obtained by use of these curred within microsomal vesicles and compounds with caution. degradation of the hormone to acid-soluRecent experiments using a variety of ble products was essentially stopped at cells and tissues indicate that complex inreduced temperature (120°C). Possible tracellular routes, in general, may be incauses for this inhibition could be (1) nec- volved in directing endocytosed materials essary hydrolytic enzymes may not be cat- to their ultimate destination within the alytically active at these temperatures; or cell (41, 42, 50). For example, Herzog has (2) endocytosis, vesicle movement, or a fu- observed in both the exocrine pancreas sion-dependent process necessary for in- (43) and epithelial cells from isolated thyput of insulin into the degradative comroid follicles (44) that certain substances partment are not functional below 20°C. go directly to the lysosomes, while others More conclusive results on what intracelare intermediately located within the Golgi lular sites are involved in insulin catabocisternae. His work has shown that the lism should be obtainable by further au- type of molecule taken up (e.g., its charge), toradiographic studies with cold perfused the method of uptake (pinocytosis or livers (27). phagocytosis, coated pits or not), as well Treatment of the perfused liver with as the cell type, can affect the route and chloroquine resulted in a significant ac- destination of engulfed substances. A varicumulation of ‘251-insulin radioactivity in able intracellular route may also be apthe tissue (Table I). The accumulation was plicable to insulin during its catabolism. in the lysosome-rich fraction indicating The breakdown of insulin has been sugthat lysosomes are involved in the deg- gested to involve the cell surface (16), radation of insulin. By gel filtration analGolgi apparatus (21), the soluble part of ysis on Sephadex G-50 65% of this labeled the cell (15), endoplasmic reticulum (40), material was found to have the same mo- and lysosomes (8,19). The fact that several lecular weight as intact insulin. Less than insulin receptors and/or affinity states for 7% of the radioactivity was small molecthose receptors exist (45) could allow the ular weight, while the remaining 28% hormone to be targeted to a variety of sites eluted in the void volume of the column. within the cell dependent upon the initial Large-molecular-weight aggregates have receptor to which it had bound. Since no been observed to elute from Sephadex G- second messenger has been determined for 50 by other scientists who have suggested insulin and it is not certain that all interthey may also be degradation products of nalized insulin is degraded completely to insulin (37). Chloroquine and other lyso- amino acids, the subcellular destination somotropic substances have now been re- of either the intact molecule or some of its ported to inhibit receptor-mediated deg- partial degradation products could be imradation of insulin in several cell types portant for the hormone’s action. For ex(20, 23-26). However, in all cases the in- ample, insulin rapidly inhibits autophagic hibition has not been substantial enough processes in liver (46-48) which appear to to argue conclusively that only a lysosomal involve membranes of both the smooth pathway of insulin degradation takes place. endoplasmic reticulum and Golgi (GERL), Our results with the perfused liver again and eventually the lysosomes (33,46). The support the idea that at least some l?reported interactions of insulin with these insulin is processed through the lyso- multiple regions of hepatocytes, therefore, somes. However, we still do not know could be related to the mechanism by whether this is the major degradative which it inhibits autophagy.
DENNIS
Al !iD ARONSON
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The
Effects
of Low Temperature and Chloroquine on ‘251-lnsulin Degradation by the Perfused Rat Liver’
PATRICIA Prmm
AND BIOPHYSICS pp. 1’70-176, 1981
A. DENNIS
NATHAN
AND
N. ARONSON,
in Biodwmistry, Althouse Laboratory, The Penns@ania Unhrsit~ Park, Pennsylvania 16802 Received
June
State
JR. University,
28, 1981
Low temperature and the lysosomotropic agent, chloroquine, were used to study the degradation of ?-insulin in a perfused rat liver. Insulin (1.5 X 10-$&i) was removed from the perfusate at 35°C with a Tllz of 12 min, and this process was slowed to 35 min at a temperature of 17°C. Essentially no degradation of ‘=I-insulin took place in the liver at 17°C. After 90 min at that temperature 64% of the liver radioactivity had accumulated in the microsomal fraction of the tissue homogenate, while at 35°C 60% of the radioactive material was in the supernatant fraction. Greater than 80% of the supernatant radioactivity was acid soluble. Rapid warming of a 17°C-treated liver to 35°C allowed the accumulated ‘=I-insulin in the microsomal fraction to be degraded to acid-soluble products in the normal manner. Chloroquine (0.2 mM) also caused the liver to degrade insulin more slowly. At 60 min after adding lzI-insulin to the chloroquine-treated liver, 50% of the radioactivity in the tissue was still present in the lysosome-rich fraction of the homogenate, while less than 10% was in this fraction in a control liver. The effects of low temperature show transfer of insulin to its degradative site is rate limiting for hormone catabolism and the inhibition by chloroquine suggests lysosomes have a role in insulin degradation by the liver.
A major site of insulin degradation in the body is the liver (1, 2). The hormone is initially bound by specific receptors on the cell surface, then becomes internalized and subsequently digested. Although these events have been demonstrated in the liver in viva (3, 4), in the perfused organ (5, 6), and in isolated hepatocytes (7, 8), as well as in other cell types (g-12), there is uncertainty regarding the exact intracellular location(s) of insulin breakdown. Insulindegrading systems have been found in the microsomal (13), cytoplasmic (14,15), and plasma membrane fractions (16) of liver homogenates, and lysosomal hydrolases obtained from this tissue are capable of rapidly digesting the peptide hormone in vitro (17, 18). Biochemical and electron 1 This work was supported by Grant from the National Institutes of Arthritis, lism, and Digestive Diseases of the United lic Health Service. 0003-9861/81/130170-07$02.00/o Copyright (B 1981 by Academic All rights
of reproduction
Preu. Inc. in any form reserved.
AM-15465 MetaboState Pub170
microscopic evidence have suggested several possible routes for the internal processing and catabolism of insulin: (a) a degradative pathway via lysosomes that is implicated in both hepatocytes and adipocytes (3, 19, 20); (b) an initial interaction with the Golgi apparatus as demonstrated in the liver (4, 21, 22); and (c) an association of the hormone with the nucleus and endoplasmic reticulum of cultured lymphocytes (11). In this current work we have studied the effects of lysosomotropic agents and low temperature on the metabolism of ‘%I-insulin by the perfused rat liver in order to substantiate that lysosomes are important for physiological degradation of this hormone. Lysosomotropic agents are compounds which accumulate in lysosomes and disrupt their functioning either by inhibiting specific hydrolytic enzymes or by changing the intralysosomal environment. These compounds have been reported to
EFFECTS OF TEMPERATURE
AND CHLGRGQUINE
partially decrease the breakdown of insulin in hepatocytes (23), adipocytes (20, 24,25), and fibroblasts (26). Mortimore and Tietze (5) several years ago reported that the normal degradation of ‘Y-insulin by a perfused rat liver could be prevented by maintaining the tissue at a cold temperature (5°C). Possibly relevant to their observation, our laboratory has recently shown that a perfused liver at 20°C stopped degrading endocytosed ‘2SI-asialofetuin (27). This glycoprotein enters hepatic lysosomes after its receptor-mediated uptake by the liver, and inhibition of its degradation at 20°C was caused by the failure of pinocytic vesicles carrying the glycoprotein to fuse with the lysosomes. Insulin that becomes internalized after binding to its receptor may also be prevented from entering the lysosomes by a similar inhibition at some critical low temperature. The experiments reported here using lysosomotropic substances and low temperature indicate that lysosomes do participate in insulin degradation by the liver, but other intracellular routes for catabolism of this important pancreatic hormone cannot be excluded. EXPERIMENTAL
PROCEDURES
Preparation of ‘25I-labeled insulin Porcine insulin (Sigma Chemical Co., 24 units/mg) was iodinated by a modification of the chloramine T method of Terris and Steiner (8). The reaction was done at room temperature in 0.1 ml of 0.3 M sodium phosphate buffer, pH 7.0. Insulin (2.0-5.0 nmol) and 1.5 mCi of carrierfree Na? (1’7 Ci/mg, New England Nuclear Corp.) were reacted upon addition of chloramine T (Eastman Kodak) equivalent to the nanomoles of insulin. After 3 to 3.5 min, 2 nmol of sodium metabisulfite/ nmol of chloramine T was added to stop the reaction, which was then mixed with 0.3 ml of 4% bovine serum albumin and run through a column (1.5 x 5.0 cm) of Sephadex G-25. The column was preequilibrated and eluted with 0.01 M Tris-HCl buffer, pH 7.0, that contained 0.05~ NaCl and 0.25% bovine serum albumin. The eluted ‘WI-insulin was placed in dialysis tubing (Spectrum Medical Industries, Inc.) and dialyzed overnight in the cold against three changes of 0.1 M sodium phosphate buffer, pH 7.0. From 95 to 98% of the radioactivity in the iodinated protein (20-70 &i/pg) both precipitated with acid (see below) and comigrated with an insulin standard on polyacrylamide gels run in sodium dodecyl sulfate
ON INSULIN
DEGRADATION
171
buffer (23). Unlabeled insulin was used to dilute this product to a final specific radioactivity of 3 to 4 &i/m. !Fre&wnt of livera Rat livers were perfused cyclically at various temperatures as previously described (27) except 2% bovine serum albumin (Fraction V, Miles Laboratory) replaced polyvinylpyrrolidone in the perfusion medium (29). Fractionation of liver homogenates, including the isolation of purified lysosomes from Tribn-treated animals (30), was according to the procedures of de Duve and his colleagues (31). The molecular weight properties of the radioactivity within subcellular fractions was analyzed on a Sephadex G-50 column (1 X 50 cm) according to the method of Terris and Steiner (8). In vitro &&w&s qf ‘ysI-insulin Digestion of ‘%Iinsulin by purified lysosomal extracts (29) was done at 37°C in 0.6 ml of 0.1 M sodium acetate buffer, pH 5.0, containing 1.0 mlb reduced glutathione. In each experiment 1 H of insulin substrate was treated with 0.155 mg of protein from the lysosomal extract. At timed intervals 20-~1 samples of the reaction were diluted with 180 pl of 0.1 116phosphate buffer, pH 7.0, that contained 1% bovine serum albumin, and 1.0 ml of cold 4% phosphotungstic acid in 2 N HCl was added to precipitate the remaining ‘l-insulin. The precipitate was centrifuged and washed once with 1.0 ml of the acid solution. The radioactivity in separate samples of the combined supernatants and the redissolved precipitate (1.0 ml of 1.0 NNaOH) was measured in a Beckman Gamma 7030 spectrometer. Analytical procedures. Protein was assayed according to the method of Miller (32) using bovine serum albumin as a standard. Chloroquine and various substrates for enzyme assays were obtained from Sigma Chemical Company. Leupeptin was purchased from Protein Research Foundation (Minohshi, Osaka, Japan). RESULTS
Effects of temperature on rndabolh of 1e51-insdin by the perfused liver. The me-
tabolism of ‘%I-insulin by the perfused rat liver was examined over the temperature range from 5 to 35°C. At 35°C the removal of ‘%I-insulin (1.0 Fg) from the per&sate occurred with a Tl12 of 12 min (Fig. 1). Acid-soluble products began to appear in the perfusate 5 to 10 min after adding the protein, and by 1 h of perfusion almost complete release of liver radioactivity was attained. Liver uptake of lSI-insulin slowed progressively at lower temperatures; for example, the Tlj2 for removal was 35 min at 17°C. A more striking effect on insulin
172
DENNIS AND ARONSON
FIG. 1. Uptake and degradation of ‘?-insulin by the perfused liver at 35°C. Livers were perfused at 35’C for 30 min after addition of 1.0 1(8 of ‘?-insulin. Samples of perfusate (0.2 ml) were removed at various times and subjected to acid precipitation as described under Experimental Procedures. Acid-precipitable radioactivity (0); acid-soluble radioactivity (0). The acid-precipitable data were corrected for the amount (25-3056) of precipitable radioactivity which was never cleared from the perfusate. This acid-precipitable material may represent the ‘“I-insulin not recognized by the liver due to damage from the iodination procedure (5).
degradation occurred over this range of temperature (Fig. 2). Only 10% destruction of the added lSI-insulin took place after 90 min of perfusion at 17°C. This
small amount of acid-soluble products appears to be the minimum level of hormone breakdown that occurs in this experimental system for the same value was obtained at 5°C. In order to determine which cellular compartments were involved in the processing of ‘%I-insulin, radioactivity was measured in subcellular fractions isolated from liver homogenates that had been prepared after perfusing the tissue for 90 min at various temperatures. Most of the radioactivity was found in the microsomal fraction at 17’C (64% of the liver radioactivity), while at higher temperature the radioactive material in the liver shifted into the soluble fraction of the homogenate (60% at 35”C, Fig. 3). A parallel release of radioactivity from both the liver itself and the microsomal fraction occurred over this experimental range of temperature, while only a small amount of the homogenate radioactivity was ever located in the nuclear (7-13%), mitochondrial (2-6%), or lysosomal (3-10s) fraction. Preliminary examination of autoradiographs obtained from a liver that had been perfused 90 min at 17°C with ‘2SI-insulin has shown that at least half of the silver grains that formed were located intracellularly. The remainder of the silver grains were still present along hepatocyte surfaces. Thus, the accumulated microsomal radioactivity at 17°C apparently
70r------l
FIG. 2. Effect of temperature on the degradation of ‘l-insulin by the perfused liver. Livers were equilibrated at the indicated temperatures and then exposed to 1.0 pg of ‘“I-insulin for 30 min. At that time 500 pg of native insulin were added for 5 min to release any labeled insulin still bound to surface receptors. The livers were then perfused briefly with cold 0.25~ sucrose, homogenized, and subjected to subcellular fractionation. The combined acid-soluble radioactivity in the perfusate and liver homogenate was measured (0) and used to detemine overall degradation of the added insulin.
FIG. 3. Effect of temperature on subcellular distribution of radioactivity in the perfused liver. Livers were treated as described in Fig. 2. Total radioactivity in both the homogenate and each of its subcellular fractions was measured: microsomal fraction, (0); final supernatant, (0).
EFFECTS
OF TEMPERATURE
AND CHLOROQUINE
FIG. 4. Reversal of the cold temperature effect on ?-insulin metabolism by the perfused liver. A liver was equilibrated at 17°C and perfused with 0.1 gg of ‘“I-insulin for 30 min. At that time (light arrow) 500 c(g of native insulin was added for 30 min to remove surface bound ‘%I-insulin. At 130 min (heavy arrow) the liver was rapidly warmed to 35°C by switching to a new, prewarmed perfusion medium. Samples of perfusate (0.2 ml) were removed at the indicated times and their content of either acid-precipitable radioactivity at 1’7’C (0) or acid-soluble radioactivity at 35°C (0) was measured.
represents both internalized and surfacebound ‘251-insulin. The latter material is not easily exchanged by large amounts of unlabeled insulin (see Fig. 4). Our laboratory previously had shown that rewarming a cold perfused liver to 35°C reestablished the normal degradation of endocytosed asialofetuin (27). Such a reversal experiment was done using ‘%Iinsulin (Fig. 4). The liver took up greater than 65% of a O.l-pg sample of radioactive insulin after 90 min of perfusion at 17°C. Adding 500 .gg of native insulin at that time caused the organ to release 28% of its previously accumulated radioactivity. Rapid equilibration of the liver to 35°C allowed normal degradation of this accumulated material. Greater than 80% of the radioactivity released into the perfusate after warm-up to 35°C was acid soluble. Effect of lysosomotropic compwtmd.s on x251-insulin degradation Leupeptin, a thiol proteinase inhibitor, has been shown to be very effective in slowing the hepatic degradation of endocytosed asialofetuin (29). However, this peptide did not alter the
ON INSULIN
DEGRADATION
173
uptake and digestion of 1.0 pg ‘ZI-insulin by the liver during 90 min of perfusion at 35°C (data not shown). Inhibition was also not observed when reactions were done in vitro using 10T3 M leupeptin and purified lysosomal extracts (Fig. 5). However, Fig. 5 does show that lysosomal breakdown of insulin to acid-soluble products was pH dependent. Hydrolysis was much reduced at pH 6.8 compared to pH 5.0. On the basis of this result we tested the effects of chloroquine and ammonium ion on insulin metabolism by the perfused liver, since these substances have been shown by others to increase lysosomal pH (34, 35) and to inhibit insulin catabolism in adipocytes (20, 24, 25), hepatocytes (23), and fibroblasts (26). Chloroquine slowed the degradation of insulin by the liver (Table I). Compared with control livers this compound caused a threefold greater amount of radioactivity to be left in the tissue 60 min after adding ‘?-insulin to the perfusate, and twice as much of the accumulated radioactivity could be precipitated with acid. NH,+ caused similar, but somewhat reduced, changes (data not shown). The subcellular distribution of radioactivity was determined for a chlo-
FIG. 5. Effects of leupeptin and pH on the digestion of ‘l-insulin by lysosomal extracts in vitro. Incubations were done as decribed under Experimental Procedures under varying experimental conditions. Aliquots were removed at the indicated times and immediately subjected to acid precipitation. The amount of acid-soluble radioactive products formed is expressed as a percentage of the total acid-precipitable radioactivity present in the ‘l-insulin substrate at 0 min: control digest at pH 5.0, (0); pH 6.0, (m); pH 6.8, (A); lo-’ M leupeptin at pH 5.0, (0).
174
DENNIS AND
ARONSON
TABLE
I
EFFECT OF CHLOROQUINE ON 12SI-I~~~~~ METAEWLISM IN THE PERFUSED LIVERY Radioactivity
in the liver
Subcellular
Treatment Control (‘“I-insulin Chloroquine
of liver only)
Percentage of injected
Percentage acid soluble
8.0 21.4
41.8 17.4
distribution
(o/o of homogenate)
Nuclear
Heavy and light mitocbondrial
Microsomal
13.2 11.8
7.0 48.8
36.0 20.5
Supernatant 44.1 18.7
’ Livers were pretreated with 0.2 mM chloroquine for 60 min prior to addition of 1.0 pg of ‘“I-insulin. After a further 60 min of perfusion the livers were homogenized and subjected to subcellular fractionation. Acid precipitation of samples (0.2 ml) of each liver homogenate was done as described under Experimental Procedures. The data shown are the average values obtained from two experiments.
roquine-treated liver after 60 min of perfusion with ‘%I-insulin (Table I). Chloroquine caused a buildup of radioactivity within the lysosome-rich fraction, ML. The chloroquine-treated liver contained 21% of the injected insulin radioactivity at that time, while the control liver contained only 8%. Neither chloroquine nor NH,+ affected the rate of uptake of *=Iinsulin by the perfused liver. DISCUSSION
Our laboratory has recently shown that temperature can be useful for studying the process of receptor-mediated degradation of asialoglycoproteins by the perfused liver (27). Catabolism ,of ‘251-asialofetuin was found to cease below 20°C and this effect was caused by a specific inhibition of the fusion of endocytic vesicles with lysosomes. The temperature studies we have now done in the perfused liver with ?insulin show this peptide hormone to have a similar, but not exact, behavior as asialofetuin. Insulin degradation was maximal between 30 and 35”C, but diminished rapidly from 27 to 20°C (Fig. 2). As already shown for asialofetuin, 1251-insulin accumulated within the microsomes at the lower temperatures (Fig. 3). In a liver perfused at 17”C, 94% of this microsomal radioactivity was acid pxecipitable and greater than 33% eluted from gel filtration columns the same as intact insulin.
Almost 50% of this insulin appears assoicated with plasma membrane even though a large amount of unlabeled insulin had been added to the perfusate prior to homogenization (unpublished autoradiographic data). The remaining radioactive material was intracellular and may be associated with pinocytic vesicles, or with other subcellular membrane components (Golgi, endoplasmic reticulum). Ward and Mortimore (36) have recently reported the isolation of vesicles containing intact insulin from a liver perfused at 22°C. These vesicles had a similar density to lysosomes, but unlike the lysosomes, did not accumulate injected Triton WR-1339. Possibly these insulin containing structures are enlarged pinocyt-ic vesicles. The nearly total inhibition of insulin breakdown observed at 17°C could be reversed by warming the liver to 35°C (Fig. 4). As the liver temperature was raised toward physiological the radioactivity shifted from its microsomal location to the supernatant fraction of the homogenate (Fig. 3). The radioactive material in the sol.uble fraction was found to be mainly degradation products with greater than 90% eluting in the small-molecular-weight fraction from a Sephadex G-50column. No significant intermediate accumulation of radioactivity was seen in any other subeellular fraction. Thus, hydrolysis of insulin within the actual degradative com-
EFFECTS
OF TEMPERATURE
AND
CHLOROQUINE
ON INSULIN
DEGRADATION
175
partment and release of its products into pathway in hepatocytes. Recent evidence the cytoplasm were both rapid steps relalso suggests that lysosomotropic agents ative to transfer of the hormone from the (chloroquine, ammonium ion) can disrupt cell surface or vesicles into the catabolic other subcellular events, such as memsite. Whatever cellular structures are in- brane recycling (38) and fusion of vesicles volved directly in insulin breakdown, the (39, 49). It is, therefore, important to inrate-limiting step for this metabolism oc- terpret results obtained by use of these curred within microsomal vesicles and compounds with caution. degradation of the hormone to acid-soluRecent experiments using a variety of ble products was essentially stopped at cells and tissues indicate that complex inreduced temperature (120°C). Possible tracellular routes, in general, may be incauses for this inhibition could be (1) nec- volved in directing endocytosed materials essary hydrolytic enzymes may not be cat- to their ultimate destination within the alytically active at these temperatures; or cell (41, 42, 50). For example, Herzog has (2) endocytosis, vesicle movement, or a fu- observed in both the exocrine pancreas sion-dependent process necessary for in- (43) and epithelial cells from isolated thyput of insulin into the degradative comroid follicles (44) that certain substances partment are not functional below 20°C. go directly to the lysosomes, while others More conclusive results on what intracelare intermediately located within the Golgi lular sites are involved in insulin catabocisternae. His work has shown that the lism should be obtainable by further au- type of molecule taken up (e.g., its charge), toradiographic studies with cold perfused the method of uptake (pinocytosis or livers (27). phagocytosis, coated pits or not), as well Treatment of the perfused liver with as the cell type, can affect the route and chloroquine resulted in a significant ac- destination of engulfed substances. A varicumulation of ‘251-insulin radioactivity in able intracellular route may also be apthe tissue (Table I). The accumulation was plicable to insulin during its catabolism. in the lysosome-rich fraction indicating The breakdown of insulin has been sugthat lysosomes are involved in the deg- gested to involve the cell surface (16), radation of insulin. By gel filtration analGolgi apparatus (21), the soluble part of ysis on Sephadex G-50 65% of this labeled the cell (15), endoplasmic reticulum (40), material was found to have the same mo- and lysosomes (8,19). The fact that several lecular weight as intact insulin. Less than insulin receptors and/or affinity states for 7% of the radioactivity was small molecthose receptors exist (45) could allow the ular weight, while the remaining 28% hormone to be targeted to a variety of sites eluted in the void volume of the column. within the cell dependent upon the initial Large-molecular-weight aggregates have receptor to which it had bound. Since no been observed to elute from Sephadex G- second messenger has been determined for 50 by other scientists who have suggested insulin and it is not certain that all interthey may also be degradation products of nalized insulin is degraded completely to insulin (37). Chloroquine and other lyso- amino acids, the subcellular destination somotropic substances have now been re- of either the intact molecule or some of its ported to inhibit receptor-mediated deg- partial degradation products could be imradation of insulin in several cell types portant for the hormone’s action. For ex(20, 23-26). However, in all cases the in- ample, insulin rapidly inhibits autophagic hibition has not been substantial enough processes in liver (46-48) which appear to to argue conclusively that only a lysosomal involve membranes of both the smooth pathway of insulin degradation takes place. endoplasmic reticulum and Golgi (GERL), Our results with the perfused liver again and eventually the lysosomes (33,46). The support the idea that at least some l?reported interactions of insulin with these insulin is processed through the lyso- multiple regions of hepatocytes, therefore, somes. However, we still do not know could be related to the mechanism by whether this is the major degradative which it inhibits autophagy.
DENNIS
Al !iD ARONSON
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