biological activity of proinsulin and related polypeptides in the fat tissue

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 248, No. 11, Issue of June 10, pp. 37533761, 1973

Printed in U.S.A.

Biological Activity of Proinsulin and Related Polypeptides in the Fat Tissue*

(Received for publication, December 18, 1972)

STEPHEN S. Yu$ AND ABBAS E. KITABCHI

From the Laboratories of Endocrinology and Metabolism, Research Xervice, Veterans Administration Hospital and Departments of Biochemistry and Medicine, University of Tennessee Medical Units, Memphis, Tennessee 38104

SUMMARY

A number of insulin-like and proinsulin-like polypeptides were employed in the study of the structure-activity relationship of proinsulin. Glucose incorporation into CO2 and lipids, and the antilipolytic activity, were used as parameters to measure the biological activity of these polypeptides. The split proinsulin and the long A chain proinsulin, with 2 connecting amino acid residues, Arg-Arg, missing, have little more activity over that of native proinsulin. The long B chain proinsulin, with the absence of 2 connecting amino acid residues, Lys-Arg, has a 3- to 4-fold increase in activity compared to proinsulin. Further removal of amino acid residues from proinsulin molecule beyond Lys-Arg level can progressively increase proinsulin activity. These studies suggest that the decreased biological activity of proinsulin may be due essentially to the blocking of the A chain of insulin part in proinsulin molecule rather than the blocking of B chain.

Insulin, with 1 or 2 arginine residues attached to COOH- terminal of B chain, loses 60% biological activity when compared to insulin molecule. The effect of positively charged groups may alter the insulin conformation or decrease the binding capacity between the insulin derivatives and the insulin receptor site, or both.

C-peptide, a by-product in the proinsulin conversion to insulin, shows no insulin-like activity in homologous and heterologous fat tissues. No antagonistic or potentiating effect of C-peptide for insulin or proinsulin can be demonstrated in homologous and heterologous fat tissues. The feedback control of C-peptide on insulin or proinsulin action is not likely to exist in the adipose tissue.

In the studies of Steiner and his co-workers (1, 2), it has been established that insulin is biosynthesised via a single chain pre-

* This work was supported in part by Veterans Administration Institutional Research Funds and by Grant AM 15509 from Na- tional Institute of Arthritis, Metabolism and Digestive Disease, National Institutes of Health, Bethesda, Maryland.

f Presented in nart as a nartial fulfillment for the degree of Doctor of Philosophy, Department of Biochemistry, College of Basic Medical Science, University of Tennessee Medical Units, Memphis, Tennessee.

cursor, proinsulin. The amino acid sequences of this prohormone have been elucidated from pancreatic tissues of different species (3-6). In addition to insulin, the presence of proinsulin and C-peptide have been demonstrated in peripheral circulation (7). Furthermore, other degradative intermediates of bovine proinsulin have been reported by Steiner et al. (8).

Many studies on the biological properties of proinsulin have been reported which suggest that this prohormone has significantly less biological activity than insulin (9-13). Previous studies from this laboratory with the isolated fat cell indicated that the biological effect of proinsulin on the conversion of glucose into COZ and lipids was only 10% as much as that of insulin. This effect was direct and not dependent upon the prior conversion of proinsulin to insulin (10). The relationship of proinsulin structure to its lower biological activity, as compared to insulin, is not understood. To gain some insight into the relationship between the structure and the biological activity of proinsulin, various polypeptide derivatives of proinsulin,’ from which certain amino acid residues have been removed, were tested for biological activity in the fat cell system. Earlier, Chance (14) has indicated similar findings for some of these polypeptides. In addition, since the significance of C-peptide in circulation is not apparent (15), C-peptides from both pork and beef were studied in heterologous and homologous fat tissues alone and in combination with insulin or proinsulin to deter- mine whether C-peptide exerts any regulatory role in the action of insulin in the fat tissue. Our studies indicate that C-peptide

1 Porcine “single component” insulin is the major purified insulin which exhibits single band on polyacrylamide disc gel electrophoresis. Monoarginine and diarginine insulins refer to insulin with Arggl and Argsl-Argaz, respectively. Desalanine insulin is the insulin lacking residue alanine at B30. Porcine desoctapeptide insulin is the insulin molecule minus 8 amino acid residues from the COOH-terminal of B chain. Porcine desglycine- desphenylalanine and desalanine-desasparagine insulins refer to the insulin lacking Glvnl-Phenl and Alaaso-Asnnl. resnectivelv. Porcine C-peptide is the connecting peptide from residues 33 to 6. Cleaved proinsulin refers to proinsulin cleaved at Leusa-Alas5 position. Desdipeptide proinsulin is the proinsulin with Lys6a- Arg,, missing. Desnonapeptide proinsulin refers to proinsulin with residues B5~--63 absent. Destridecapeptide proinsulin is the proinsulin with residues B~1--63 missing. Bovine Intermediate I refers to bovine proinsulin lacking residues Lys59-Args0. Bovine Intermediate II refers to proinsulin with Argai-Argaa missing. Bovine C-peptide is the connecting peptide from residues 33 to 58.

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has no significant effect in the adipose tissue. Preliminary counted by the method of Gliemann (20). The mean count of results of this work have been reported earlier (16, 17). at least four independent dilutions was used as cell number.

EXPERIMENTAL PROCEDURE

A&n&-Male Holtzman rats weighing 110 to 120 g were used for glucose metabolism experiments. The rats were fed Purina lab chow and had access to food and water up to the time of death. Calf and pork adipose fat pads were freshly obtained from the local slaughter house.

Chemicals and Media-Bovine plasma albumin (Fraction V, Lot G-34405) was purchased from Armour Pharmaceutical Company, Chicago, Ill. Albumin was dialyzed for 24 hours, changed three times against Krebs-Ringer bicarbonate buffer, adjusted to an albumin concentration of 20% with Krebs-Ringer bicarbonate buffer and kept frozen in aliquots. For experimental use, one vial of albumin was diluted with Krebs-Ringer bicarbonate buffer, pH 7.4, to give a final solution with albumin concentration of 4%. This 4% albumin Krebs-Ringer bicarbonate buffer was the buffer used throughout these experiments unless specified otherwise.

Porcine “single component” insulin, monoarginine, diarginine, desalanine insulins, C-peptide, as well as proinsulin and proinsulin intermediates-split, desdipeptide, desnonapeptide, and destridecapeptide proinsulins-were gifts of Dr. R. Chance of Eli Lilly Company, Indianapolis, Ind. Bovine insulin, proinsulin, proinsulin intermediates I and II, and bovine C-peptide were supplied by Dr. D. F. Steiner of the University of Chicago. Chemical definition and source of these compounds have been reported elsewhere (14, 17). pi-Z4 corticotropin, used as a lipolytic reagent, was offered by Dr. H. Strade of Organon Lab- oratories. [U-i4C]Glucose was obtained from New England Nuclear Corp. All of the other chemicals were reagent grade and purchased commercially.

Preparation of Isolated Fat Cells-Fat cells were prepared by the modified (18) method of Rodbell (19) and Gliemann (20) as follows. Holtzman male rats, which had access to food and water up to the time of death, were killed by decapitation. The epididymal fat pads were immediately removed. The fat pads of three or four rats were pooled and cut into small segments of 5 to 10 mg. Fat tissue, 700 to 1000 mg, was placed in a plastic vial containing 5 mg of collagenase in 2 ml of 4% albumin Krebs- Ringer bicarbonate buffer. This digestion was carried out in a Dubnoff shaker at 120 cpm for 35 min at 37”. At the end of the digestion period, collagenase contamination was washed out by diluting cells with 16 ml of buffer and centrifuging at 500 rpm for 2 min. Buffer was removed from beneath cell layer with plastic tubing and syringe. Three to four washings were re- quired for each cell preparation. The washed cells were filtered through one layer of silk organza to remove any large pieces of capillaries. For glucose incorporation studies, the cell suspension was diluted to a volume containing approximately 20 mg of fat cell per ml. A l-ml aliquot of the pooled isolated fat cells was poured into the plastic vial in which [U-WJglucose (200,000 cpm) with final glucose concentrations of 0.55 mM and proinsulin or related polypeptides of various concentrations had already been added. For antilipolytic experiments, a l-ml cell suspension of 40 mg of fat cell was added to each plastic vial with various concentrations of polypeptides and 0.15 pg of Dim24 corticotropin. The fir-24 corticotropin, used as the lipolytic agent, was dissolved in vehicle of 0.5y0 albumin in saline, pH 3.5. Corticotropin was added to the incubation medium immediately after the incubation started. The fat cells were

Preparation of Fat Pieces-Calf and pork fat pads from thigh portion were immediately removed after animal was killed. The fat pad was cut into 25- to 50.mg segments. Pooled fat pieces were weighed on a torsion balance. Fat pieces, 250 mg, were added to the incubation vial containing glucose concentration 0.55 mM with [UJ4C]glucose (200,000 cpm) and various testing materials as indicated by the procedure of Bray (21).

All of the vials were gassed for 1 min with 95% 02-5s CO? at the start of incubation. Incubation was performed at 37” in the Dubnoff shaker at a speed of 50 cpm. For glucose uptake studies, the reaction was stopped by dumping 0.5 ml of 8 N HzS04 into the incubation medium at the end of 2 hours of incubation. In glycerol studies, the experiments were terminated by pouring the incubation medium into a prechilled tube after 1 hour of incubation. Fat cells were aspirated from the chilled incubation mixture after being centrifuged at 1000 rpm for 2 min in a re- frigerator centrifuge.

Method of Assay-For glucose incorporation studies, 14C02 was collected on the strips of the filter paper saturated with hydroxide of Hyamines by the method of Gliemann (20). W in the total lipids was extracted with hexane according to the method of Rodbell (19). Results were calculated as nanoatoms of glucose carbon incorporated per lo5 cells in 2 hours of incubation. Glycerol was determined by a fluorimetric method of Chernick (22). Results were expressed as nanomoles of glycerol released per lo5 cells in 1 hour of incubation.

RESULTS

Biological Activity of Bovine Insulin, Proinsulin, and Inter- mediates I and II-An anionic trypsin-like enzyme that con- verts proinsulin to insulin has been isolated from bovine pancreas by Yip (23). This enzyme preparation cleaves the bond between Gly*r and Argso and that between Alamo and Arg,,. Kemmler et al. (24) found that two peptide bonds could also be cleaved by a combination of pancreatic trypsin and carboxy- peptidase B in v&-o to yield Intermediate I with residues Lysss- Arg’g60 missing and Intermediate II with residues Argsi-Arg32 absent from bovine proinsulin molecule. These Intermediates I and II are believed to be the obligatory intermediate forms in the conversion process. The biological activity of these polypeptides was measured in the isolated fat cells from the rat. The results of these studies for glucose conversion to COz and lipids are shown on Figs. 1 and 2, respectively. The data were plotted on a semilogarithmic scale. The maximal rate of conversion for these compounds was found to be the same. Table I summarizes the relative potency of all of the compounds used in Figs. 1 and 2. The values were derived from the concentration of half-maximal response. As can be seen, bovine proinsulin requires a concentration 10 times that of insulin to obtain the same response. Intermediate I, the long B chain derivative of proinsulin, activates proinsulin activity a-fold. On the other hand, Intermediate II, the long A chain derivative of proinsulin, cannot significantly activate proinsulin. A mixture of Inter- mediates I and II, with a ratio of 40:60 originally isolated by Steiner, has the biological activity between these two intermediates.

Biological Activity of Porcine Proinsulin-like Polypeptides- Several proinsulin derivatives, with C-peptide moiety split or partially removed, have recently been isolated by Chance (14). They are valuable for the structure-activity comparison with proinsulin to elucidate the mechanism of activation of proinsulin.


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A BEEF INTERMEDIATE I

+ BEEF INTERMEDIATE x a xt

. BEEF INTERMEDIATE IL

Beef Proinsulin

FIG. 1. Dose-response curves for bovine insulin, proinsulin, Intermediate I, Intermediate II, and the mixture of Intermediates I and II on the conversion of glucose to CO* in the isolated fat cell from rat. The experimental procedure is described in the text. Each point represents an average of six experiments ZIZ the standard error of the mean.

e s B

700 I I I .BEEF INSULIN

a h .A 1 ABEEF INTERMEDIATE I 5 ; 600 zx t

+W6b;TERMEDlATE 1 B=

MOLAR CONCENTRATION

FIG. 2. Dose-response curvesof bovine insulin, proinsulin, Inter- mediate I, Intermediate II, and the mixture of Intermediates I and II on the conversion of glucose to lipids in the isolated fat cell from rat. Each point represents the average of six experiments f the standard error of the mean.

Using the conversion of [UJ4C]glucose into 14C02 and [14C]lipide as parameters of activity, the dose-response curves of these intermediates were studied and results are shown in Figs. 3 and 4. Porcine proinsulin with cleavage at Leu54-Alas5 has slightly increased biological activity when compared with proinsulin. Desdipeptide proinsulin with Lys&rgs3 absent shows a a-fold increase over the biological activity of proinsulin. Further removal of amino acids from C-peptide part beyond desdipeptide

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TABLE I Biological activity of bovine insulin, proinsulin, and intermediates

in isolaled fat cells

Compounds

Insulin. ............ Proinsulin. .......... Intermediate I ....... Intermediate II. ..... Intermediates I and

II (40:60) .........

- I

Half-maximal AC- WSpOIlSe tiv-

concentration ity

%

3.6 X lo-” M 100 3.1 x lo-” M 12 1.1 x lo- M 33 2.1 x 10-rOM 17

1.3 x 10-iOM 28

Glucose conversion to lipids

Half-maximal b%pOllSe

concentration

AC- tivity

3.5 x 10-10 M 3.2 X lo-” M

1.2 x 10-lOM

2.3 X lo-” M

%

100 11 30 15

1.5 x 10-M 23

-

-

Destridecapeptld Proinsulin

6.4 i IO“' M

\

n PROINSULIN

+PORCINE DESNONAPEPTIDE PROINSULIN

.PORcINE DESDIPEPTIDE

PROINSULIN

.PORc,NE SPLIT PROINSULIN fl= zz 0 *PORCINE PROINSULIN

IQ” IO-‘0 10-9 10-a

MOLAR CONCENTRATION

FIG. 3. Dose-response curves of porcine proinsulin and proinsulin-like intermediates on the glucose conversion to CO9 in the isolated fat cells from rats. Each point represents the average of six experiments f the standard error of the mean.

-. ".,_,..- ,.._"_,.. + PORCINE DESTRIDECAPEPTIDE

PROINSULIN

. PORCINE DESNONAPEPTIDE PROlNSULlN

. PORCINE DESDIPEPTIDE PROINSULIN

. PORclNE SPLIT PROINSULIN

*wXlrlMc PRnlNSIII IN

MOLAR CONCENTRATION

FIG. 4. Dose-response curves of porcine proinsulin and proinsulin-like intermediates on the conversion of glucose to lipids in the isolated fat cell from rats. Each point represents the average of six experiments rt the standard error of the mean.


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TABLE II E

Biological activity o.f porcine proinsulin and proinsulin-like inter- 3 700, I I I I 1 Gliates 7% isolated fat ceils

Half-maximal l-t%pO”Se

concentration

AC- Half-maximal AC- tiv- tiv- ity

WSpO”Se concentration ity

-

3.5 x lo-” M 3.0 x lo-” M

2.5 X lo-lo M

%

100

11 14

3.4 x lo-” M

3.3 x lo-” M

2.7 X lo-‘O M

%

100 10 12

9.8 x lo-” M 36 1.0 X lO+nI 34

9.1 x lo-” M 39 9.2 x lo-” M

8.4 X lo-” M 42 8.4 x 10-l’ M

37 If

41

Compounds

Insulin. Proinsulin. Split proinsulin..

Desdipeptide proinsulin

Desnonapeptide proinsulin.

Destridecapeptide.

FIG. 6. Dose-response curves of porcine insulin and insulin-like derivatives on the conversion of glucose to lipids in the isolated fat cells from rat. Each point represents the average of six experiments f the standard error of the mean.

T‘\BLE III

Biological activity of porcine insulin and insulin-like derivatives in isolated fat cells

Glucoseec~gwsion 2

Glucose conversion to lipids

AC- tivity

%

100 40 39

97

3

3

1

Compounds Half-maximal AC- Half-maximal

XSpO”Se tiv- concentration

response ity concentration

%

Insulin 3.5 X lo-” M 100 3.4 X lo-” M

Monoarginine insulin. 9 .O X lo-” M 39 8.5 x lo-” M

I N

FIG. 5. Dose-response curves of porcine insulin and insulin-like derivatives on the conversion of glucose to CO* in the isolated fat cell from rat. Each point represents the average of six experiments f the standard error of the mean.

38 8.7 X lo-” M

95 3.5 x lo-” M

3 1.2 x 10-9 M

Diarginine insulin. 9.2 X lo-” M

Desalanine insulin. 3.7 X 10-I1 M

Des GlyA,-des PheRl insulin. 1.3 X lO-9 M

Des AlaBao-des AsnAn, insulin. 1.5 X 1om9 M

Desoctapeptide insulin. 5.4 X 10m9 M

2 1.3 x 10-g M level will progressively increase proinsulin activity. Thus, removal of 13 amino acids from the amino end of A chain (destridecapeptide) increases biological activity to that of 41% of insulin. The comparative values of these polypeptides are given in Table II.

1 4.4 x 10-Q M

T.U~LE IV

Biological Activity of Porcine Insulin-like Polypeptides-The presence of small amounts of monoarginine and diarginine insulin

Antilipolytic activity of porcine insulin, proiusulin, am! related polypeptides in isolated fat cells11

can be detected in the commercial insulin preparation. Arginyl insulin might be the unfinished product during the conversion of proinsulin in the islets of Langerhans as proposed by Kemmler et al. (24). The concentration-response curves of arginyl insulin and other insulin-like polypeptides on the conversion of glucose to CO* and lipids are shown in Figs. 5 and 6. The monoarginine and diarginine insulins have only 400/, biological activity compared with insulin. The des Alasso-des AsnA21, des G1yA1-des PheB1, and desoctapeptide insulins show 37, or less insulin activity. The half-maximal response concentrations of the above compounds are given in Table III.

Antilipolytic Activity of Porcine Insulin-like and Proinsulin-like

Antilipolytic effect half-maximal re-

iponse concentrationb Compounds Activity

% Insulin-like

Insulin Monoarginine insulin.

Diarginine insulin. Desalanine insulin.

Proinsulin-like Proinsulin. Split proinsulin.

...... ......

1.7 X lo-” M 100

3.8 x lo-” M 44 4.0 x 10-l’ M 42 1.7 x lo-” M 100

1.8 x lo-‘O M 1.6 X lo-l0 M

4.5 x lo-” M

4.3 x lo-” M

4.0 x lo-” M

9 11

38 40 42

Polypepticles-The direct biological activity of these polypep- Desdipeptide proinsulin.

tides in the isolated fat cell system can be confirmed by using Desnonapeptide proinsulin..

yet another parameter, inhibition of lipolysis. The antilipolytic Destridecapeptide proinsulin..

activity of these insulin-like and proinsulin-like polypeptides studied on the isolated fat cells with &H corticotropin as the lip-

0 ~~-24 corticotropin (1.5 pg per ml) was used as the lipolytic

olytic agent is shown on Table IV. The antilipolytic activity agent.

6 The half-maximal response concentration was obtained from of these polypeptides is correlated very well with the glucose dose-response (glycerol release-hormone concentration) curve.

oxidation activity; i.e. arginyl insulins have 40% biological The result is the average of six observations.


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TABLE V

Effect of porcine insulin, proinsulin, and C-peptide on [U-W]glucose conversion to ‘VT02 on bovine, porcine, and rat fat tissuea

Preparation

Control...... Insulin. Insulin.

Insulin. Proinsulin. Proinsulin. Proinsulin.

C-peptide. C-Peptide

. .

Concentration

I 4.0 x 10-l’ M

1.0 x 1@‘o M

2.0 x 1o-‘o M

3.0 x 10-O M

1.0 x l(P M

2.0 x lo-9 M

4.0 x lo-‘M

4.0 x 10-M

-

_-

-

Rat fat cells

natoms ‘CO* rel.%wd/lO~ cells/Z hrs

40.1 f 3.6b 142.9 f 12.1

210.5 f 13.6 234.8 f 13.4 140.2 f 8.2

227.3 f 17.8 42.6 f 4.2 43.4 f 4.6

- - Bovine fat pieces

n&??m WY02 released/g lissue/Z hrs

420.5 f 85.2

705.1 f 24.8 904.5 f 86.8

599.9 f 54.8

414.8 f 104.8 454.2 f 69.8

Porcine fat pieces

natomr ‘CO2 wlcased/g tissue/Z ts

631.5 f 39.2

1313.1 f 56.2

1555.3 f 54.6

945.4 f 47.6

684.0 f 8.6 665.0 f 40.0

a Method of assay is described in the test.

b Each value is the average of three observations f standard error of the mean.

TABLE VI

Effect of bovine insulin, proinsulin, and C-peptide on [U-W]glucose incorporation into WO2 on bovine, porcine, and rat fat tissues

Preparation

Control......

Insulin. Insulin. Insulin. Proinsulin. Proinsulin. Proinsulin. C-Peptide

C-Peptide.

T -.

Concentration

5.0 x lo-” M

1.0 x 1o-‘o M

2.0 x lo-” M

3.0 x lo-lo M

1.0 x lo+ M

2.0 x lo-* M

4.0 x I@* M

4.0 x 10-M

Rat fat cells

na‘oms ‘CO2 releasexi/lO’ cells/Z hrs

35.2 f 1.9@

176.6 f 11.4 210.5 f 11.8

240.4 f 6.5 140.3 f 8.2 227.4 f 9.8 242.4 f 9.6

37.0 f 3.0 33.4 f 3.8

Bovine fat pieces

natas “CO* released/g tissue/Z hrs

420.5 f 85.2

586.0 f 65.6

865.5 f 115.6

584.7 f 75.8 804.0 f 94.6

430.5 f 80.2 420.0 f 76.8

Porcine fat pieces

natoms “CO, released/g tissue/Z hrs

631.5 f 39.2

1152.4 f 41.0

1502.6 f 52.4

1070.9 f 20.0

1571.0 f 137.6 681.9 f 47.0 657.9 f 29.0

a Each value is the average of three observations f standard error of the mean.

strength, compared to that of native insulin. Split proinsulin

has the same biological activity as proinsulin. Desdipeptide, desnonapeptide, and destridecapeptide proinsulins have biological activity three to four times that of intact proinsulin.

E$ect of Porcine Insulin, Proinsulin, and C-peptide on Glucose Metabolism in Beef, Pork, and Rat Fat Tissues-C-peptide, an obligatory by-product during insulin biosynthesis, is present in circulation in equal molar concentration with insulin (7, 8). No biological activity of C-peptide has been demonstrated in the isolated fat cell system; however, most studies were carried out by using bovine or porcine C-peptide in rat tissue (10-13). Due to the large variation in amino acid sequence of C-peptides isolated from different mammalian pancreas tissues, Oyer et al. suggested (25) the possibility that C-peptide might have biological activity in homologous fat tissue. To study this phe- nomenon, the porcine C-peptide, as well as insulin and proinsulin, was assayed on homologous and heterologous fat tissues. Data shown on Table V indicate that at high concentration, porcine insulin and proinsulin enhance glucose uptake 6-fold in isolated fat cells and l-fold in beef and pork fat pieces. However, porcine C-peptide shows no significant stimulation on glucose osidation in any of three animal fat tissues, including pork fat pieces.

Effect of Bovine Insulin, Pro&z&n, and C-peptide on Glucose Metabolism in Homologous and Heterologous Fat Tissues-Bovine insulin, proinsulin, and C-peptide were studied in beef, pork,

and rat fat tissues. The results are shown in Table VI. Bovine insulin and proinsulin, similar to that of porcine, have a stim- ulatory effect on homologous and heterologous fat ti sues, whereas the C-peptide shows no biological activity on these three different fat tissues.

Effect of C-peptide on Biological Activities of Insulin and Pro- insulin-Previous experiments have shown that neither bovine nor porcine C-peptide affects the insulin and proinsulin action on the isolated rat fat cells (10). To investigate the possibility that bovine or porcine C-peptide have regulatory function on insulin or proinsulin activity in homologous fat tissues, these compounds were studied in combination with insulin or proinsulin in the rat and porcine fat tissues. The results summarized in Table VII suggest that porcine and bovine insulin or proinsulin, with or without homologous C-peptide, show no biological activity changes in homologous or heterologous fat tissues.

Antilipolytic Effect of Porcine Insulin, Proinsulin, and C-peptide in Isolated Fat Cells-Lipolysis, induced by /II-Z4 corticotropin at a concentration approaching maximal response, is inhibited by insulin and proinsulin but not by C-peptide (11). Results summarized in Table VIII indicate that the combination of C-peptide with insulin or proinsulin has no synergistic or inhibitory effect on the biological activity of insulin or proinsulin per se. Insulin, proinsulin, or C-peptide alone show little or no lipolytic activity.


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E$ect of C-peptide on biological activity of insulin and proinsulin.

Preparation

Control Porcine C-pep-

tide........... Porcine insulin.. Bovine insulin. Porcine proin-

sulin Bovine proin-

snlin. Bovine C-peptide

and bovine in- sIllin,

Porcine C-peptide and porcine insulin.

Bovine C-pep-, tide and bovine proinsulin.

Porcine C-peptide and porcine proinsulin

_-

‘

-

Concentration

1.0 x lo-’ M

1.0 x 10-10 M 1 .o x 10-10 M

1.0 x lo- M

1.0 x lo+ M

Rat fat cells

36.8 f 4.7”

35.0 f 4.0 236.7 f 13.2

245.6 f 14.6

247.6 & 12.0

244.1 z!r 15.4

239.5 f 16.6

-

-

Porcine fat pieces

na1oms "CO1 reieaseailOj cells/Z tars

631.5 f 39.2

665.0 f 40.1 1312.1 f 56.2

945.4 f 47.6

1302.2 f 54.0

955.6 f 39.6

a Each value is the average of three observations f standard error ofmthe mean.

l-

DISCUSSION

An earlier report from this laboratory (10) suggested that proinsulin has intrinsic biological activity based on the failure of Kunitz pancreatic trypsin inhibitor and trasylol to inhibit proinsulin activity, as well as the lack of a lag period of proinsulin transformation into insulin. Recently, Narahara (26) also showed that proinsulin has an intrinsic biological effect on frog sartorius muscle. The biological activity of proinsulin is about 10% that of insulin by different parameters (8, 9, 11).

In the proinsulin molecule, it is likely that the C-peptide is the steric hindrance that decreases the binding affinity of the insulin moiety of proinsulin to the insulin receptor. The proinsulin structure has been studied by many authors. The primary structures of porcine and bovine proinsulin are depicted in Figs. 7 and 8. Franks and Veros (27, 28)) based on the similar self-association characteristics and ultra-absorption pattern between insulin and proinsulin, proposed that the insulin moiety of proinsulin molecule has a similar conformation to that of the native insulin. Furthermore, Franks and his co-workers (29) showed that the insulin moiety in split and desnonapeptide proinsulins is essentially the same conformation as the free insulin molecule. The C-peptide, with a high content of proline, exists in a random coil form (28, 30). The insulin structure postulated by Blundell et al. (31) indicated that the A chain of the molecule is a compact unit around which the B chain is wrapped. The 3 amino acid residues As, As, A 10, varying most among mammalian species, were believed to be part of the antigenic deter- minant and directed out from the surface of the molecule. Res- idues A1, As, Alg, and A21 were thought to be on the surface of the insulin molecule and not involved in the aggregation of dimer

TABLE VIII

Effect of porcine C-peptide on corticotropin-induced lipolysis of isolated fat cell

Base-line .............. p-,-24 corticotropm (0.15

pg/ml) .............. +Insulin ............ +Insulin. ............ +Proinsulin ........... +Proinsulin. ........... +C-peptide. .......... +C-peptide. ........... + Insulin. ............ +Insulin ............. +Insulin ............. +Insulin ............. +Insulin ............ +Proinsulin. ........... +Proinsulin. ........ Insulin. .......... ... Insulin. ............ Proinsulin. ......... ... Proinsulin. ............. C-peptide .............. C-peptide .............. Insulin. .............. Insulin. ............... Proinsulin. ............ Proinsulin. .............

Concentration

4.0 x 10-l’ M

1.0 x 1w9 M

3.0 x lo-‘o M

1.0 x lo-* M

4.0 x lo-’ M

4.0 x lo-‘M

4.0 x lo-” M

1.0 x 10-M

4.0 x lo-” M

4.0 x lo-” M

1.0 x lo* M

3.0 x lo-‘O M

1.0 x l@* M

4.0 x lo-” M

1.0 x 10+ M 3.0 x 10-l’ M

1.0 X 10-8~

4.0 x lo-* M

4.0 x lo-’ M

4.0 x lo-” M

1.0 x 10-M

3.0 x lo-lo M

1.0 x lo-* M

Addition

Proinsulin 3.0 x lo-lo M Proinsulin 1.0 X 1O-8 M

Proinsulin 1.0 x lo- M

C-peptide 4.0 x IO-7 M

C-peptide 4.0 x lo- M

C-peptide 4.0 x lo-‘M

C-peptide 4.0 x lo-’ M

C-peptide 4.0 x lo-7 M

C-peptide 4.0 x 10-7 M C-peptide 4.0 x lo+ M

C-peptide 4.0 x lo-‘M

,J Values are the average f the standard error of triplicate determinations.

Concentration --

Lipolysis

650.7 + 24.0 215.9 f 1.0 113.2 +Z 7.0 257.9 f 7.4

99.2 f 14.4 612.8 f 19.6 580.4 f 28.0 112.3 zt 27.6

146.6 f 14.0 124.9 * 16.2

199.3 + 39.8 130.8 zt 41.6 220.9 z!z 7.8 133.9 + 7.2

6.1 zk 1.4 10.8 f 5.4 10.1 f 1.4 13.5 h 2.8

11.5 + 9.4 7.4 f 4.0

10.1 + 1.4

6.8 f 5.4 6.8 f 5.4 9.5 * 0.1

-

wioles glj’cerol rdeasea/los cells/hr

10.4 zk 2.4~


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FIG. 7. Primary structure of porcine proinsulin as shadecl circles.

PORCINE PROINSULIN proposed by Chance (14). The connecting peptide moiety is depictEd by

BOVINE FIG. 8. Amino acid sequence of bovine proinsulin as proposed

portion of proinsulin.

or hexamer (32). Further study of the chain folding of insulin by Mercola et al. suggested that the insulin in neutral solution is similar to the insulin in the crystalline state (33). As proinsulin and insulin aggregate in a very similar way, C-peptide cannot be on any of the surfaces involved in aggregation. It is possible that the C-peptide is looped around the insulin moiety of proinsulin and is arranged on the outside surface of insulin hexamer. The central part of the C-peptide has a high content of glycine residues surrounded by largely nonpolar residues. This amino acid sequence suggests that the central part of the molecule is more flexible and may bend toward the insulin moiety through the interaction of the nonpolar side chain (4). The space between the glycine A1 residue and alanine BzO residue is equivalent in length to 3 amino acid residues apart (34). The

PROINSULIN by Nolan et al. (4). Shaded circles designate connecting peptide

two dipeptides Arg-Arg and Lys-Arg, through which C-peptide is hooked to the insulin molecule, are positively charged at physiological pH. Due to the electrostatic interaction, these two dipeptides might repel each other to give enough room to ac- commodate the insertion of C-peptide.

In the bovine yroinsulin Intermediate I and porcine desdipeptide proinsulin, the 2 basic connecting residues, Lys-Arg, are missing. These two polypeptides have a long B chain and partially Eree A chain because the C-peptide is still in the vicinity of the NHZ-terminal of A chain or covering part of the A chain. InFigs. 1 and 2, the biological activity of bovine insulin, proinsulin, and Intermediates I and II all exhibit sigmoid dose-response curves on the semilogarithmic scale. These polypeptides reach maximal and half-maximal response at different concentrations.


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3760

The relative ability of these compounds to convert [ U-14C]glucose into i4C02 and [‘%]lipids is depicted in Table I with the following degree of potency: insulin > Intermediate I > Intermediate II > proinsulin. It is clear that the deblocking of proinsulin A chain, as that of Intermediate I, can activate proinsulin activity a-fold. The removal of connecting dipeptide from the B chain terminal has little activation effect. The similar results of the activity of porcine proinsulin, insulin, and related polypeptides are shown in Figs. 3 and 4. The porcine split proinsulin, with both A chain and B chain blocked by cleaved segments of C-peptide, has slightly increased biological activity when compared to proinsulin. The porcine desdipeptide proinsulin, structurally analogous to bovine Intermediate I, can activate proinsulin activity 3-fold. Desnonapeptide and destridecapeptide proinsulin progressively increase in biological activity over that of desdipeptide proinsulin. The removal of 2 basic connecting residues, Lys-Arg, from the NHz-terminal of A chain is thought to be a basic requirement for proinsulin activation in both bovine and porcine proinsulin. The NHz-terminal of C-peptide of desdipeptide proinsulin is a flexible coil and partially overlaps the NHz-terminal of A chain or certain sections of A chain. As more and more amino acid residues are removed beyond desdipeptide level, the amino end of A chain is under less and less influence of the rest of C-peptide and becomes a “relatively free” A chain terminal. This correlates well with a progressive increase in the biological activity of desnonapeptide anddestrideca- peptide proinsulins.

The des Alanso-des AsnAzi and des Gly,,-des Phem insulins in isolated fat cell assay, as also shown elsewhere (35), exhibit only 3% or less insulin activity. Carpenter (36) showed that des Alasa insulin has the full biological potency, as that of insulin. Brandenburg (37) demonstrated that the des Phenl insulin is as active as native insulin. This suggests that both of the terminal residues of B chain are relatively less important for insulin activity. On the other hand, the 2 terminal residues of A chain, glycine at A1 and asparagine at A2i, are relatively important for hormone activity. The acetylation studies of insulin by Lindsay and Shall (38) showed that the acetylation of glycine*l amino group, phenylalanineBl amino group, or the e-amino group of the lysineBzs will not affect the biological activity of insulin. However, the substitution at the glycine*i amino group by the larger residue, acetoacetyl or thiazolidine- carbonyl, produced a decrease in biological potency. Modifica- tion of lysineg~~ or phenylalaninesl amino groups with larger chemicals such as Sepharose (39) or dextran (40) does not affect the insulin activity. The structure of insulin as elucidated by crystallography (34) suggests that both GlyAl and AsnAzI are on the surface of the molecule. Freychet el al. (42), using isolated fat cell and a liver plasma membrane radio receptor system, found that the reduced biological activity of porcine proinsulin, bovine desoctapeptide insulin, and bovine des Alanso- des AsnAzi insulin was attributed to the reduced affinity of these compounds for the receptor. These studies suggest that the NH?- and COOH-terminal of A chain GlyAl and AsnAt may participate in the binding of insulin to its receptor or have the stabilization effect in the region of insulin “active site.” The proinsulin with glycineA1 terminal blocked by C-peptide is possibly a major reason for the 90% loss of activity when compared to insulin.

formational change in diarginine insulin in addition to the 2 amino acid residues added (29). It is possible that the con- formational change in diarginine and possibly monoarginine insulins may interfere with the binding of insulin to its receptor on the cell membrane. In Table IV, the biological activity of porcine insulin, proinsulin, and related polypeptides was confirmed with another parameter, antilipolysis. All of these compounds tested showed antilipolytic effect against /3imZ4 corticotropin-induced lipolysis with the same relative strength as glucose oxidation studies. Studies by Chance (14) on mouse convulsion assay with some of these porcine polypeptides suggest that biological activity for proinsulin and split proinsulin is around 20% that of insulin. Desdipeptide and desnonapeptide proinsulins, as well as monoarginyl and diarginyl insulins, show around 60% activity compared to that of native insulin. The activity of these polypeptides in mouse convulsion assay is higher than that in the isolated fat cell assay. This difference might be due to either a prolonged half-life of these compounds in the body (41) or the degradation of these polypeptides to a biologically more potent compound in circulation.

Although the presence of C-peptide has been demonstrated in circulation in equimolar concentration with insulin, lack of its biological effect on isolated fat cells (10-12) of heterologous tissue has now been confirmed and extended to homologous fat tissues. In addition, lack of an antagonistic or potentiating effect of C-peptide on insulin or proinsulin in rat fat cell has now been extended to homologous tissues. Due to a large sequence variation of C-peptide among various species, it has been implied that C-peptide might be species-specific and exhibit a regulatory function on homologous rather than heterologous tissues. The comparative effect of bovine C-peptide in Table V and porcine C-peptide in Table VI with insulin and proinsulin on the adipose tissue of beef and pork, as well as the isolated fat cells of rat, were studied. These studies demonstrate that C-peptide has no insulin-like activity in heterologous or homologous fat tissues. Furthermore, the effect of combination of C-peptide of beef or pork with homologous and heterologous insulin or proinsulin were studied in porcine fat pieces and rat fat cells. The data shown in Table VII exhibit no antagonistic or synergistic effect of C-peptide on insulin or proinsulin. Similarly, the lack of antilipolytic property of porcine C-peptide on the rat fat cell, alone or in combination with insulin and proinsulin, is demonstrated in Table VIII. It would thus appear unlikely that C-peptide exerts any significant biological effect on insulin-sensi- tive tissue such as adipose tissue.

Table III and Figs. 5 and 6 show that the blocking of COOH- terminal of B chain with monoarginine or diarginine results in a 60% decrease in insulin activity. Circular dichroic spectra studies by Franks and his associates show that there is con-

Acknowledgmenls-The authors wish to thank Drs. R. Chance, D. Steiner, and H. Strade for their generous supply of chemicals used in these studies. The editorial assistance of Mrs. E. Salemi is gratefully acknowledged.

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Stephen S. Yu and Abbas E. KitabchiBiological Activity of Proinsulin and Related Polypeptides in the Fat Tissue

1973, 248:3753-3761.J. Biol. Chem.

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