intermediates by collagenase proteinases adsorbed to isolated ... - NCBI

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show that isolated fat cells can degrade proinsulin to intermediates due to their contamination with ... Recently human proinsulin, producedby recombinant.

Biochem. J. (1988) 255, 277-284 (Printed in Great Britain)

277

Conversion of biosynthetic human proinsulin to partially cleaved intermediates by collagenase proteinases adsorbed to isolated rat adipocytes William C. DUCKWORTH,*§ Daniel E. PEAVY,t Frederick G. HAMEL,* Juris LIEPNIEKS,t Mary R. BRUNNER,t Richard E. HEINEYt and Bruce H. Frankt

*Veterans Administration Medical Center and the Department of Medicine, University of Nebraska Medical School, Omaha, NE 68105, tVeterans Administration Medical Center and the Department of Physiology, Indiana University School of Medicine, and the $Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, U.S.A.

Studies of the biological activity of proinsulin have resulted in widely varying conclusions. Relative to insulin, the biological activity of proinsulin has been reported from less than 1 00 to almost 200. Many of the assays in vitro for the biological potency of proinsulin have utilized isolated rat adipocytes. To examine further the interaction of proinsulin with rat adipocytes, we prepared specifically-labelled proinsulin isomers that were iodinated on tyrosine residues corresponding to the A14, A19, B16 or B26 residue of insulin. These were incubated with rat adipocytes and their metabolism was examined by trichloroacetic acid precipitation, by Sephadex G-50 chromatography, and by h.p.l.c. chromatography. By trichloroacetic acid-precipitation assay, there was little or no proinsulin degradation. By G-50 chromatography and subsequent h.p.l.c. analysis, however, we found that the labelled proinsulin isomers were converted rapidly and almost completely to materials which eluted differently on h.p.l.c. from intact proinsulin. This conversion was due primarily to proteolytic activity which adsorbed to the fat cells from the crude collagenase used to isolate the cells. Two primary conversion intermediates were found: one with a cleavage at residues 23-24 of proinsulin (the B-chain region of insulin), and one at residues 55-56 in the connecting peptide region. These intermediates had receptor binding properties equivalent to or less than intact proinsulin. These findings show that isolated fat cells can degrade proinsulin to intermediates due to their contamination with proteolytic activity from the collagenase used in their preparation. Thus the previously reported range in biological activities of proinsulin in fat cells may have arisen from such protease contamination. Finally, the present findings demonstrate that a sensitive assay for degradation of hormones is required to examine biological activities in isolated cells.

INTRODUCTION Proinsulin is the single chain precursor of insulin [1]. Within the pancreatic B cell, proinsulin is converted to insulin by proteolytic removal of the connecting peptide region resulting in the loss of two pairs of basic residues and the production of insulin and C peptide [2]. Not all of the proinsulin is converted to insulin, however. The pancreas also releases intact proinsulin into the circulation and significant amounts of proinsulin are present in blood, especially during fasting [3]. The physiological role of circulating proinsulin remains uncertain. Many studies have been performed with proinsulin extracted from animal pancreas [4-12] but their variable results have prevented firm conclusions being drawn. Recently human proinsulin, produced by recombinant DNA techniques, has become available for examination [13]. This material is homogeneous by h.p.l.c. analysis and has been examined for biological activity in vivo and in vitro. The results, however, have varied from study to study. Proinsulin in vivo appears to have 15-20 o of the activity of insulin on a molar basis, but interpretation of the results is complicated by the longer half-life of § To whom reprint requests should be addressed.

Vol. 255

proinsulin and the multiplicity of tissues affected [14]. Studies in vitro have shown much less activity for proinsulin compared with insulin. We have reported proinsulin to have 1-3% of the activity of insulin as measured by binding to isolated fat cells and to liver cell membranes, and by glucose incorporation into lipid in isolated fat cells [15,16]. Other laboratories have reported that in isolated fat cells, proinsulin has 3-10% of the activity of insulin [17,18]. To examine these differences further, we have studied the degradation of proinsulin by isolated fat cells by analysing on h.p.l.c. proinsulin incubated with adipocytes. We have found that these cells rapidly and almost completely convert proinsulin to intermediate forms. These intermediate forms have been characterized and appear to be novel forms of split proinsulin.

EXPERIMENTAL Animals Male Sprague-Dawley rats were obtained from Harlan Industries, Indianapolis, IN, U.S.A. Animals

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were given free access to water and a commercial pellet diet and were housed in a controlled environment with a 12 h light-12 h dark cycle for at least 5 days before use. Only rats weighing 150-200 g were selected for use in these studies. Fat cell isolation Adipocytes were prepared from epididymal fat pads by a modification [19] of the collagenase-digestion procedure of Rodbell [20]. Krebs-Ringer/Hepes buffer, pH 7.4, containing 4 % bovine serum albumin and 0.55 mM-glucose was used in all isolation and incubation steps. During this preparation the cells were washed 4 times with fresh buffer and then were preincubated for 30-60 min. The preincubation buffer was then replaced with fresh 37 °C buffer for the experimental incubation. The preincubation step has been found, in previous studies, to remove any insulin-degrading activity present at the end of the cell washes. Insulin and proinsulin iodination Porcine single-site [125Iliodo(A14)insulin (sp. act. 300340 1Ci/1tg) was prepared and isolated as described previously [21]. The preparation of human [125I]_ monoiodoproinsulin (sp. act. 210-230 ,uCi/,ug) and proinsulin derivatives in general used the same methodology as for the preparation of the iodoinsulin. For the purposes of clarity and consistency, we chose to identify the iodination site in proinsulin and proinsulin intermediates by the terminology used for insulin. Thus proinsulin iodinated on the tyrosine-79 residue is referred to as [125I]iodo(A14)proinsulin since this residue becomes the A14 amino acid after conversion of proinsulin to insulin. H.p.l.c. Characterization of proinsulin and its degradation products was performed using a Beckman Model 332 gradient-liquid chromatograph fitted with a 0.46 cm x 25 cm Zorbax (150A) C8 column and a heater. After injection of a 100 ,l sample, the eluant was derived from solvents A and B using a 72 min gradient from 26 % to 29 % CH3CN in 0.25 M-sulphate, pH 2.0, at 45 °C and 0.9 ml/min flow rate. The effluent was collected at 1.0 min intervals with a Gilson Micro Fractionator. Radioactivity was counted using a Searle Model 1185 automatic gamma system counter. Degradation products of proinsulin were isolated by an extension of the same h.p.l.c. system using a 0.5 ml sample loop and 40 °C column temperature, with a 70 min gradient from 26.4% to 27.4% CH3CN at 1.0 ml/min flow rate. Materials Collagenase (type I, lot 40P080 or 43D289) was purchased from Worthington Biochemicals, Freehold, NJ, U.S.A. Pentex bovine serum albumin (fraction V) was from Miles Biochemicals, Elkhart, Indiana, U.S.A., and was exhaustively dialysed before use as described previously [19]. D-[2-3H]Glucose was purchased from Amersham Corp., Arlington Heights, IL, U.S.A. Crystalline porcine insulin (lot 615-07J-256 or 615-2H2-300) was provided by Dr. R. E. Chance of the Eli Lilly Research Laboratories, Indianapolis, IN, U.S.A. Biosynthetic human proinsulin was prepared as previously described [16].

Proinsulin solutions were freshly prepared by dissolving the contents of vials that contained calibrated quantities of human proinsulin. The protein content of the vials had previously been determined by dissolving contents in 0.01 M-HCI and then subjecting aliquots to amino acid analysis and to h.p.l.c. analysis. The protein, content was calculated from the results of the amino acid analyses and agreed with results of a u.v. determination of protein content. Freshly-prepared solutions of insulin were calibrated by u.v. determination. RESULTS Each of the four specifically-labelled monoiodoproinsulin isomers was incubated with isolated fat cells for 60 min at 37 'C. Aliquots taken for trichloroacetic acid solubility showed that less than 2 % of each of the labels was soluble in 10 % (w/v) trichloroacetic acid at the end of 60 min of incubation. Each of the incubation media was chromatographed on Sephadex G-50 with results as shown in Fig. 1. The A14, A19 and B16 isomers eluted as a single broad peak and the B26 isomer eluted as two distinct peaks, one in the region corresponding to proinsulin and one in the small-molecular-mass fractions. The A14, A19, B16 and B26 proinsulin regions were pooled into a front half and a back half. The smallmolecular-mass B26 peak was also collected separately yielding nine different pools of fractions. Each of these was lyophilized, resuspended, and injected on h.p.l.c.

1251-Proinsulin 1251 Insulin

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3' c x

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2' 1

0

44

56

68

80 92 Fraction no.

104

116

Fig. 1. Sephadex G-50 elution patterns of 1 251]iodoproinsulin incubated with isolated fat cells Specifically-labelled proinsulin isomers were incubated with isolated fat cells and the incubation medium chromatographed on a Sephadex G-50 column in I Macetic acid. The elution profiles of A14, A19 and B16 isomers were identical and are shown by the dashed line. The elution pattern of the B26 isomer is shown by the light continuous line whereas the starting materials (A 14, Al9, B16, and B26) are shown by the heavy line. For the A 14, A19 and B16 isomers the fractions eluting in the position of proinsulin were collected and divided into two pools, a front half (fractions 68-80) and a back half (fractions 81-90). For the B26 isomer, three pools were collected, a front half (fractions 66-74), a back half (fractions 75-82), and a smaller-molecular-mass pool (fractions 83-100).

1988

Cleavage of human proinsulin by collagenase adsorbed to adipocytes 80

279 80 -

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Fig. 2. H.p.l.c.-elution profile of '25Iliodo(A14)proinsulin incubated with isolated fat cells [l25l]Iodo(A14)proinsulin was incubated with isolated rat adipocytes and the Sephadex G-50 separated materials chromatographed on h.p.l.c. The starting material is shown in panel (a). The front half of the G-50 peak (fractions 68-80, Fig. 1) is shown in panel (b) and the back half (fractions 81-90, Fig. 1) in panel (c).

Figs. 2-4 show the results with A14, A19 and B16. The control proinsulin elution pattern is given in panel (a). Panel (b) shows the front half of the Sephadex G-50 column from the incubation medium and panel (c) the back half of the Sephadex G-50 profile. As can be seen in panel (b), relatively little intact proinsulin remained in the incubation medium, with two predominant peaks eluting between the positions of insulin and proinsulin. From the back half of the Sephadex G-50 peaks (panel c), a small amount of proinsulin, the two intermediatesized materials, and a peak of material eluting slightly earlier than insulin can be seen. Fig. 5 shows the results from the B26-proinsulin incubation. Relatively little intact proinsulin remained in the Sephadex G-50 proinsulin region and with two prominent intermediates (panels b and c) also present. The small-molecular-mass peak from the Sephadex G-50 column (panel d) eluted early from the h.p.l.c. as a single peak. These results suggest that when proinsulin is incubated with isolated fat cells, it is converted almost entirely to three major derivatives. With each of the isomers, two intermediates elute from a Sephadex G-50 column in the proinsulin region and elute from an -h.p.l.c. column in the region between proinsulin and insulin, a region where proinsulin intermediates normally elute [22]. These results suggest that these materials are proinsulin cleaved at two different sites yielding two different intermediates, but both intermediates contain all or almost all of the amino acids present in intact proinsulin. With A14, A 19, and B 16, a Vol. 255

.-I

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.

.

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Fig. 3. H.p.l.c.-elution profile of 1'25Iiodo(Al9)proinsulin incubated with isolated fat cells Conditions are as in Fig. 2.

smaller molecule, as judged by Sephadex G-50, can be identified which elutes from h.p.l.c. slightly before insulin, suggesting that a material is present with these three isomers that contains most of the structure of insulin but may be missing part or all of the connecting peptide. With B26, the generation of a small peptide suggests that 80(a)

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Fig. 4. H.p.l.c.-elution profile of 1'25IJiodo(Bl6)proinsulin incubated with isolated fat cells Conditions are as in Fig. 2.

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the portion of the proinsulin molecule which was lost contains the B26 residue and thus, may consist of a cleavage between the B19 cystine and B26, and an additional cleavage in the connecting peptide region. To determine the specificity of the proteolytic activity, [125I]iodo(A14)insulin was incubated with isolated fat cells and the medium analysed as described above. On h.p.l.c., over 90%0 of the insulin-sized labelled material from Sephadex G-50 eluted in the position of intact [125I]iodo(A14)insulin, whereas in parallel experiments using ["25I]iodo(A 14)proinsulin, only 100 of the labelled material eluting from Sephadex G-50 in the proinsulin region eluted from h.p.l.c. in the position of intact [1251]iodo(A14)proinsulin. These results support the hypothesis that the proteolytic activity in the isolated fat cell preparation attacks proinsulin in the connecting peptide portion of the molecule. The next experiment was performed to determine whether the degradation of proinsulin was due to the cells or to degrading activity in the incubation medium. Cells were prepared and incubated for 60 min in the usual manner, but without added proinsulin. The tubes were then centrifuged and the incubation medium removed. [1251]Iodo(A14)proinsulin was added to the medium and incubated at 37 °C for another 60 min. At the end of the incubation, medium was chromatographed on Sephadex G-50 and injected on h.p.l.c. as described above. The elution pattern was identical with that shown in Fig. 2. These results demonstrate that the conversion of the proinsulin to intermediates was due not to the cells, but to proteolytic activity present in the medium. To examine further the specificity of the proteolytic activity, medium from preincubated cells was incubated with ['251]iodoinsulin or with ['251]iodoglucagon. Degradation was assayed by trichloroacetic acid precipitability and by h.p.l.c. of the labelled material. Less than 20 of the [1251]iodoinsulin was degraded by this assay and on h.p.l.c., over 95 % of the labelled material eluted as intact ['25I]iodoinsulin. In contrast, after 60 min of incubation, 5000 of the [1251]iodoglucagon was converted to tri-

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Fig. 5. H.p.l.c.-elution profile of 1'25IIiodo(B26)proinsulin incubated with isolated fat cells

Panel (a) shows the starting material. Panel (b) shows the front half of the Sephadex G-50 peak (fractions 66-74, Fig. 1). Panel (c) shows the back half of the Sephadex peak (fractions 75-82, Fig. 1) and panel (d) shows the small-molecular-mass peak (fractions 83-100, Fig. 1).

Table 1. Distribution of radioactivity eluted from a Sephadex G-50 column after incubation of 1l25IIiodo(A14)proinsulin with various preparations of adipose tissue

[1251]Iodo(A14)proinsulin was incubated with various preparations of adipose tissue and then chromatographed on Sephadex

G-50 as in Fig. 1.

Radioactivity (%) No. of

High molecular

Proinsulin

Low molecular

mass

region

mass

Preparation

experiments

Incubation time (min)

Isolated fat cells Intact fat pad

4

60

2.6+0.2

95.2+0.5

1.5+0.5

3

60 120

3.0+0.6 3.6+0.4

93.5+ 1.4 92.2+0.9

3.2+0.6 4.1 +0.4

2

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22.5 21.8

29.1 18.3

47.1 59.9

2

60

8.6

80.9

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Fat pad

homogenate Fat pad

homogenate (1:8 dilution)

1988

Cleavage of human proinsulin by collagenase adsorbed to adipocytes

chloroacetic acid-soluble material. These data demonstrate that the medium from incubated adipocytes contains proteolytic activity which will readily degrade proinsulin and glucagon but not insulin. Since isolated fat cells are prepared from epididymal fat tissue by collagenase digestion, two possibilities for the origin of the proteolytic activity and the generation of these proinsulin intermediates exist. One is that fat tissue itself has enzymes present which can produce these materials, and the other is that the activity is due to residual contamination of the final cell preparation with proteases from the crude collagenase used to isolate the cells. To determine if the proinsulin-degrading activity was derived from the tissue itself, intact fat pads were incubated with ['25I]iodo(A14)proinsulin for 60 and 120 min. At each of these times, aliquots of the incubation media were removed and chromatographed on Sephadex G-50. At 60 min, 93.5 % of the radioactivity eluted in the ' proinsulin' peak with 3.2 % in the small-molecularmass peak and the remainder in the void volume (Table 1). At 120 min, the proinsulin peak was 92.2% .-and the small-molecular-mass peak 4.1 % suggesting that a small amount of proinsulin was degraded to low-molecularmass material. The 'proinsulin' peak from the Sephadex G-50 column was lyophilized and injected on h.p.l.c. Over 9500 of the labelled material eluted as intact proinsulin with no radioactivity eluting in the positions of the intermediates noted in earlier experiments. These results suggest that intact fat pads slowly degrade proinsulin, but do not form the same intermediates seen with collagenase-prepared isolated fat cells. To confirm this observation, intact fat pads were homogenized and the homogenates incubated with [1251]iodo(A14)proinsulin. Aliquots of the incubation media were chromatographed on Sephadex G-50. Incubation times were 60, 120 and 180 min. At 60 min, only 29.1 % of the starting radioactivity eluted in the proinsulin region and by 180 min, this had decreased to 18.30%, demonstrating that fat cells contain proteolytic activity which can degrade proinsulin. This experiment was repeated with a 1:8 dilution of the homogenate incubated for 60 min with [1251]iodo(A14)proinsulin at which time only 10.2 % of the labelled material eluted as small-molecular-mass material, as determined on Sephadex G-50, more comparable with that seen with intact cells. The 'proinsulin'-sized material from this experiment was lyophilized and injected on h.p.l.c. No radioactivity eluted in the positions of intermediates, suggesting that the enzymes which produce these materials are not present in fat tissues. To determine whether the collagenase used to prepare the cells could produce the intermediates, [1251]iodo(A14)proinsulin was added to several concentrations of the collagenase (10.0, 1.0, 0.1 and 0.01 utg/ml) and incubated at 37 °C for 60 min. These collagenase concentrations represented 1, 0.1, 0.01 and 0.001 o of the original concentrations of collagenase used in the fat cell isolation. At the end of the incubation, equal volumes of extraction mix were added and the tubes immediately frozen. Each incubation mixture was subsequently thawed and immediately chromatographed on a Sephadex G-50 column. With the three lower concentrations of collagenase, progressive widening of the 'proinsulin' peak was seen. With the highest concentration of collagenase (10 #g/ml), essentially all radioactivity Vol. 255

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eluted from the G-50 as small-molecular-mass material. Each of the 'proinsulin' peaks from the 0.01, 0.1 and 1 jtg/ml collagenase incubations were individually pooled, lyophilized, and analysed on h.p.l.c. With the 0.01 ,ig/ml collagenase, a very small peak of intermediate I, representing less than 5 % of the starting material, was seen. With 0.1 ,ug/ml collagenase, 19 % of the labelled material eluted in the position of intermediate I with no detectable II, and with 1 ,ug/ml, 50 % was present in the intermediate peaks, with approximately equal distribution between peaks I and II. Less than 10 % of the radioactivity remained as intact proinsulin. These results suggest that proteases in the crude collagenase could generate materials consistent with the intermediates. The next question was whether sufficient contamination of the final cell preparation could occur to explain the production of the intermediates. To answer this, the collagenase used in the isolated fat cell preparation was iodinated with 125I by lactoperoxidase to a specific activity of 31.3 ,uCi/#sg. A mixture of unlabelled collagenase and the iodinated material (2.0 mg containing 6 ,Ci radioactivity) was used to prepare isolated cells by the usual procedure. Aliquots were taken at each step to determine the radioactivity and the amount of proteolytic activity (using ['25Iliodoglucagon as substrate) remaining with the cells. In a parallel study, [14C]sucrose was added to the cell preparation procedure to determine the retention of the liquid phase throughout these various steps. The results are shown in Table 2. As can be seen, only 0.0007 % of the [14C]sucrose was carried through to the final cell incubation. In contrast, 17-times as much (0.012%) of the labelled collagenase was present in the final cell incubation. Retention of proteolytic activity was even greater with 0.24% of the total glucagondegrading activity present in the media of the final cell preparation. These results suggest adsorption of the collagenase and especially the proteolytic activity in the collagenase, to the adipose cells. The observed contamination of the final preparation was sufficient to account for the production of the intermediates. Table 2. Percentage of radioactivity and proteolytic activity remaining with cells during preparation of isolated fat cells

Isolated fat cells were prepared from rat epididymal tissue in the usual way (see Methods section) except that either 1251-labelled collagenase or [14C]sucrose-containing buffer were used. Aliquots were taken at each step to determine the percentage of the starting radioactivity remaining. Aliquots were also taken at each step and incubated with '251-glucagon to determine proteolytic activity. 125I_

[14C] Initial Wash 1 Wash 2 Wash 3 Wash 4 Pre-incubation Incubation

Sucrose

1251_ Collagenase

100 16.25 3.22 0.8 0.06 0.008 0.0007

100 32.2 14.5 7.7 4.3 0.02 0.012

Glucagondegrading

activity 100 27.0 12.1 5.9 2.7 0.8 0.24

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W. C. Duckworth and others

To determine if other collagenase-prepared cells besides adipocytes degrade proinsulin, isolated hepatocytes were prepared and incubated with [125I]iodo(A14)proinsulin. The incubation media was separated, chromatographed on Sephadex G-50 and then injected on h.p.l.c. Essentially all of the radioactivity in the proinsulin peak from G-50 eluted from h.p.l.c. in the position of intact proinsulin with no evidence of material in peaks corresponding to the intermediates. These results suggest that collagenase proteolytic activity does not adsorb to hepatocytes as it does to fat cells. The proteolytic activity in the crude collagenase was examined further by chromatography on DEAESephacel. Column fractions were assayed for proteolytic activity using [125I]iodoglucagon as substrate. The DEAE column was equilibrated in 20 mM-sodium acetate, pH 6.2, and eluted in a stepwise fashion with that buffer containing 0, 0.1, 0.2 and 0.5 M-NaCl. Each of the steps contained a peak of glucagon-degrading activity and thus each was pooled separately, and an aliquot incubated with [125I]iodo(B26)proinsulin with subsequent chromatography on Sephadex G-50. Only the activities eluting with 0.2 M and 0.5 M-NaCl contained proinsulindegrading activity with the 0.2 M peak having by far the greatest activity. The 0.2 M-NaCl peak was then chromatographed on a Sephadex G-200 column which had been calibrated with blue dextran, alcohol dehydrogenase, ovalbumin, chymotrypsinogen, and riboflavin. The column fractions were assayed for glucagon-degrading activity and a broad peak of activity extending from an apparent Mr of 25 000 to 85000 was obtained. The fractions in this peak were then assayed for proinsulin-degrading activity using the Sephadex G-50 assay. The proinsulin-degrading activity was found in the region corresponding to an apparent Mr of 30000. The DEAE-purified proteinase and an aliquot of adipocyte infranatant were incubated with [1251]_ iodo(B26)proinsuiin in the absence and presence of various group-specific inhibitors (Table 3). Degrading activity was quantified by Sephadex G-50 chromatography and measuring the amount of material in the lower-molecular-mass fraction (see Fig. 1). The chelator O-phenanthroline essentially totally inhibited proinsulindegrading activity in both preparations. Pepstatin and phenylmethanesulphonyl fluoride had partial effects amounting to less than 5000 inhibition. N-Ethylmaleimide slightly inhibited the DEAE enzyme preparation but had no inhibitory effect on the infranatant Table 3. The effect of various inhibitors on proinsulin-degrading activity of adipocyte infranatant and DEAE-purified collagenase

Inhibition (%) Inhibitor N-Ethylmaleimide Phenylmethanesulphonyl fluoride O-Phenanthroline

Pepstatin None

Concen- Adipocyte DEAEtration infranatant collagenase I

mM mM

0 25

22 51

I

mM

100

37

98 42

0

0

1

1O,ug/ml

activity. These results suggest that the primary degrading activity in these relatively crude preparations is a metalloproteinase. Cleavages in the connecting peptide region of pro-

insulin can alter the biological activity of the molecule. A close correlation between the receptor-binding characteristics and the biological activity has been shown [16]. It was therefore of interest to determine if the intermediates exhibited altered binding relative to intact proinsulin. [1251]Iodo(A14)proinsulin was incubated with isolated fat cells infranatant, chromatographed on Sephadex G-50, and the proinsulin peak injected on h.p;l.c. Radioactivity corresponding to the intermediates was collected separately, diluted and adsorbed on Sep-Paks. After washing, each intermediate was eluted with ethanol and dried. The intermediates were then redissolved in buffer and added to liver cell membranes. The intact proinsulin peak from the infranatant was examined in the same manner. After 24 h at 4 °C, the amount of radioactivity bound to the membranes was determined by centrifugation, removal of the media and counting in an autogamma counter. Non-specific binding was determined by using excess unlabelled proinsulin and was subtracted from total to yield specific binding. Taking intact proinsulin binding as 100 %, intermediate I bound 91 0 and intermediate II bound 7400, as well as the intact material. Since the binding potency of proinsulin in vitro is very low 116], it is impossible to state with certainty that the binding of the intermediates is decreased, but clearly neither intermediate showed a significantly increased binding to liver cell membranes compared with intact proinsulin (results not shown). Although cleavages in the connecting peptide region of proinsulin can yield increases in the biological activity of the molecule, the extent of the alteration varies with the location of the cleavage. For example, cleavages near the N-terminal of the A chain may greatly increase activity, whereas cleavages near the C-terminal of the B chain only slightly increase activity [16]. Cleavages in the middle of the connecting peptide may produce no changes. The results here suggest that the intermediates are not likely to have cleavages near the end of the A chain. To identify the cleavages present in the two intermediates, [125I]iodo(B26)proinsulin was incubated with isolated fat cell medium and the two intermediates separated on h.p.l.c. Each of these was sequenced in an amino acid sequenator. Intermediate II had radioactivity appearing in the third cycle and intermediate I in the 26th cycle demonstrating that intermediate II had a cleavage between Gly-23 and Phe-24, and I a cleavage after B26, presumably in the connecting peptide region. A similar study was done with A14 proinsulin and intermediate I was sequenced. Sequencing showed radioactivity present in the 24th cycle showing a cleavage in the connecting peptide region between amino acids Pro-55 and Leu-56. The small-molecular-mass component from the B26 isomer was also sequenced, showing radioactivity in the third cycle, further confirming the cleavage at B23-B24. DISCUSSION These studies show that proinsulin incubated with isolated fat cells is rapidly degraded by extracellular proteases. The primary degradation occurs through the 1988

Cleavage of human proinsulin by collagenase adsorbed to adipocytes

formation of two intermediates, one with a cleavage at B23-B24 and the other with a cleavage in the connecting peptide at C55-C56. These two intermediates are further degraded resulting in the loss of the B24-C55 region with the formation of a near insulin-sized product. Extending the incubation time of proinsulin with the conditioned medium results in more of the insulin-sized product, but relatively little degradation to smaller fragments occurs, suggesting that the insulin-sized product, like insulin itself, is relatively resistant to the extracellular proteinase. With shorter incubation times or more diluted collagenase the C55-C56 bond (forming intermediate I) is cleaved to a greater extent than the B23-B24 bond. The formation of these products occurs as a result of contamination of the adipocyte preparation of proteases from the crude collagenase used to prepare the cells. Several studies have shown that proteolytic activity is required in the collagenase preparations to prepare isolated fat cells [23,24]. Thus crude collagenase, rather than purified collagenase, is necessary for this purpose. The observation that isolated fat cells contain proteolytic activity as a contaminant from collagenase has been made previously by other investigators [23]. The contamination appears to be due to the adsorption of the proteolytic activity to the adipose cells rather than to carry over of the digesting solution to the final preparation, since additional washes did not remove the activity, and the proteolytic activity in the final preparation was greater than the amount of buffer contamination. This appears to be a particular problem with adipose cells, since collagenase-prepared hepatocytes did not have the same activity. The medium-degrading activity has no detectable activity toward insulin, illustrating the difficulty in selecting collagenase preparations to use in cell preparations. The collagenase used in this study was selected for isolated fat cell preparation after screening over 30 collagenase lots from various suppliers. The screening procedure was directed at selecting a collagenase which produced isolated fat cells which had appropriate biological responses to insulin and which had no insulin-degrading activity in the medium. In spite of the rigorous screening protocol, the use of the cells for an assay other than one involving insulin resulted in a cell preparation which was not ideal for this purpose. These results show that isolated fat cell preparations must be carefully examined before being used to draw conclusions about hormonal activity. In particular, these observations may help explain some of the variable results on proinsulin activity in adipocytes [15-18]. Other preparations of collagenase may well have differing amounts of this or other contaminating proteases, which might alter interpretation of results. For example, cleavages in the proinsulin molecule near the region of the N-terminal of A chain greatly increase the biological activity of the molecule [16]. If such cleavages occurred and were undetected, the biological activity of 'proinsulin' would appear to be much higher than in actuality. We have previously reported that the biological activity of proinsulin is only 1-3 00 that of insulin [15,16], whereas other laboratories have reported up to 100% [17], although this was subsequently revised to 3 %0 [18]. Much of the data supporting this conclusion was derived from analysis of the biological activity of proinsulin in isolated fat cells. Based on the studies described here, questions may be raised as to the previous conclusions. From consideration of the results, we feel that our Vol. 255

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previous conclusions are correct. Firstly, we obtained comparable results from studies with IM 9 lymphocytes, isolated hepatocytes, and isolated liver cell membranes, preparations which do not contain the same proinsulindegrading activity. Secondly, binding of proinsulin to adipocytes under conditions of reduced temperature where the intermediates are not formed, also showed a potency of proinsulin relative to insulin of only 1 %. Thus we feel that our previous conclusions on the biological activity of proinsulin are valid. We wish to emphasize that the most commonly used assays to determine degradation (trichloroacetic acid, gel filtration, receptor binding) would not have detected the changes seen in this study. We were able to detect the degradation only by the use of a sensitive h.p.l.c. assay. Thus conclusions on the biological activity of proinsulin (or other hormones) must be considered tentative until appropriate studies are performed to exclude the possibility of alterations in the molecule during assay. At present, h.p.l.c. appears to be the most sensitive method to determine this. This work was supported in part by Veteran's Administration Research Funds. The excellent secretarial assistance of Ms. Marianne Arp is gratefully acknowledged.

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Received 2 February 1988/20 May 1988; accepted 7 June 1988

1988