Acta histochem. 82, 35-39 (1987)

VEB Gustav Fischer Verlag Jena

Institute of Neurobiology and Brain Research, Academy of Sciences of the GDR1), Magdeburg, Institute of Anatomy2), and Department of Experimental Immunology of the Clinic of Internal

Medicine3 ), Medical Academy Magdeburg, GDR

Proteolytic degradation of insulin and glucagon in rat brain during ontogenesis4)

By MICHAEL REISER!), HANS-GERT BERNSTEIN2), SIEGFRIED ANSORGE3),

and ALFRED DORNt2)

With 3 Figures

(Received April 24, 1986)

Summary

The degradation of insulin and glucagon was investigated in rat brain, kidney, and liver dur­ing postnatal ontogenesis. It was found that a maximal activity around 13 d is unique for CNS and differs remarkably from time-course in liver and kidney. It is concluded that this result sup­ports the hypothesis of a growth promoting role of insulin in developing rat brain.

Introduction

During the last 5 years, evidence has been accumulated that an insulin-related peptide occurs in the central nervous system, which is, possibly, a product of the brain itself (HAVRANKOVA et al. 1978; DORN et al. 1983). The function of this peptide in the brain of vertebrates is as yet an unsolved problem. However, several lines of evidence suggest that insulin might playa role as a growth promoting hormone (RAI­ZADA et al. 1980 ; YANG et al. 1981).

The present study was undertaken from the point of view that synthesis and phy­siological function of insulin in nervous tissue implicate the existence of mechanisms of degradation or clearance from the site of action. Therefore, the cleavage of insulin during the first 20 d of postnatal ontogenesis was investigated. This is a period, when an influence of insulin on neuronal development is supposed (BERNSTEIN et al. 1984; KAPPY et al. ]984).

Our investigation is founded upon knowledge which was collected from insulin and glucagon degradation in other organs. So, in the liver, one of the major sites of insulin action and degradation, the initial step in insulin metabolism is thought to be the binding to specific receptors on the cell membrane, followed by internalization of the peptide-receptor complex and proteolytic cleavage of the hormone (SONNE and GLIEMANN 1983). The enzyme insulin/glucagon degrading proteinase (IGP, E.C. 3.4.22.11) has been accorded a key role in cell mediated degradation of insulin, be­cause ist accounts for most of the insulin degrading enzyme activity. lGP purified

4) Presented in part at the symposion on the occasion of the 25th anniversary of the foundation of the Institute of Anatomy at the Medical Academy Magdeburg, GDR, September 1985.

3"

36

30

20

10

M. REISER et al.

insulin and glucagon degrading activity [pkat/gwet weight]

A

_ insqlin

09luc090n

B C

A-Tris'HCL pH· 8j) B-Tris-HCL+GSH (3mmol/l) c- Tris· HCl +oPhenanthroline <1Smmoltl} 0- Tris-HCl +GSH+oPhenanthroline E- Tris-HCL+EDTA USmmo''')

-~ D E ABC D E

Fig. 1. Insulin and glucagon degrading activity in brain cytosols in the presence of EDTA, glu­tathione and o-phenanthroline.

from rat liver was characterized as a metal-dependent endopeptidase. This enzyme is located in the cytosol and is specific for both insulin and glucagon, the Km for gluca­gon being higher by an order of magnitude (ANSORGE et al. 1984).

Method Wistar rats of specified ages (3, 10, 13, 19 d; adult) were killed by decapitation. Kidney, liver,

and brain were removed and homogenized in a 10 fold volume of 0.25 molll sucrose. The homo­genates were centrifuged at 12,000 X g for 15 min. The supernatant was centrifuged at 105 X g for 60 min and the cytosol fraction was stored for determination at -20°C. The preparation of 1251_ labelled hormones was performed by the chloramine-T technique, followed by a purification on a Sephadex G 50 column.

The enzyme activities were determined by measurement of the loss of trichloroacetic acid pre­cipitable radioactivity during incubation of iodinated peptides with the cytosols. 0.1 ml of the 105 X g supernatant was mixed with 0.1 ml of Tris-buffer (pH = 8.0). After 2 min of preincubation at 37°C 0.1 ml, radioiodinated hormone (0.83 pmolll insulin; 5.58 pmolll glucagon) was added and incubation was performed at 37°C for 20 min. The reaction was terminated by cooling the sample in an ice bath and by subsequent addition of 0.1 ml serum albumin solution (20 gil) and 0.2 ml trichloroacetic acid (150 gil). The plastic tubes were centrifuged at 4 X 103 X g for 30 min. The supernatant was gently removed and the pellet was used for counting of radioactivity. Protein content was determined by Lowry method.

Results

Insulin and glucagon were degraded during incubation with cytosol fraction of the rat brain at all stages of postnatal ontogenesis. As shown in Fig. 1, this glucagon

Degradation of insulin and glucagon in rat brain

Ins!!ljn and glucagon degrading actIvIty C pkat/g wet weight]

3 to 13 19 adult days

[pkat/g protein]

brain

~insulin Dglucagon

10 13 19 adult postnatal days age

37

Fig. 2. Insulin and glucagon degrading activity in brain cytosols at several stages of postnatal development.

and insulin degrading activity could be totally inhibited by o-phenanthroline (15 mmol/ I in incubation buffer) and, partially, by EDTA (15 mmol/l). An addition of 3 mmol/l glutathione (GSH) to the incubation medium increases the insulin breakdown, where glucagon is degraded at the same amount. An incubation in buffer containing o-phen­anthroline and GSH results in a total inhibition of glucagon cleavage and an decrease of insulin breakdown to the GSH induced level.

Fig. 2 shows the degradation of the pep tides by cytosols of rat brains at several stages of postnatal ontogenesis. The activity of glucagon and insulin degradation related both to protein content of the cytosol and to the wet weight of the organ has been found to be maximal around 13th postnatal d.

The time-course of insulin and glucagon degradation during postnatal ontogenesis in liver and kidney is shown in Fig. 3. In liver, the rate of peptide degradation is approximately the same in young and adult rats. In contrary, in kidney cytosols, the enzyme activity as related to protein content differs remarkably from enzyme activity as related to wet weight.

38 M. REISER et al.

insulin and glucagon degrading activity

Cpkat/g wet weight] kidney

IIIIIIII insu lin

~glucagon

3days 10days adult

Cpkat/g protein]

liver ~insulin

c:::J glucagon

3days 10 days adult postnatal age

Fig. 3. Insulin and glucagon degrading activity In the cytosols of liver and kidney at several stages of postnatal development.

Discussion

The data on the inhibition of the insulin and glucagon degrading activity in brain cytosols are in a good agreement with findings obtained with liver and kidney cytosols (AN SORGE et a1. 1984) and make it reasonable to argue that insulin and glucagon are cleaved by an enzyme, which shares a great deal of common properties with liver IGP. However, our recent failure to show an immu­nological relationship between antibodies against liver IGP and the brain enzyme speaks against this possibility. On the other hand, at the present state of our study the participation of more than one enzyme in the cleavage of the peptides cannot be excluded. Nevertheless, from the point of view of a nerve growth promoting role played by insulin, the maximum proteolytic activity around postnatal 13th d is of interest, the more as the insulin receptor density at neuronal membranes is also maximal at this time (KAPPY et a1. 1984).

This result differs remarkably from the time-course of insulin degradation during postnatal ontogenesis of other organs. In liver, the enzyme activity remains at the same level at all stages of postnatal development, whereas in kidney, the activity of peptide degradation related to wet weight is increasing after birth to adult levels, parallely to the increasing protein content of kidney homo­genates during this period (WACHSMUTH and STOY 1976). The circumstance that insulin degra-

Degradation of insulin and glucagon in rat brain 39

dation in the brain is maximal at the same time, when the density of insulin receptors is highest (KAPPY et al. 1984) and membrane protein synthesis is also maximal, adds further support to the hypothesis of an essential role of insulin in brain maturation.

Literature

ANSORGE, S., BOHLEY, P., KIRSCHKE, H., LANGNER, J., and WIEDER ANDERS, B., The insulin and glucagon degrading proteinase of rat liver: A metal· dependent enzyme. Biomed. Bio· chim. Acta 43, 39-46 (1984).

BERNSTEIN, H.·G., DORN, A., REISER, M., and ZIEGLER, M., Cerebral insulin· like immunoreactiv· ity in rats and mice. Drastic decline during postnatal ontogenesis. Acta histochem. 74, 33 t.o 35 (1984).

DORN, A., BERNSTEIN, H.·G., RINNE, A., ZIEGLER, M., HAHN, H.·J., and ANSORGE, S., Insulin· and glucagon-like peptides in the brain. Anat. Rec. 207, 69-77 (1983).

HAVRANKOVA, J., SCHMECHEL, D., ROTH, J., and BROWNSTEIN, M., Identification of insulin in rat brain. Proc. Natl. Acad. Sci USA 75,5737-5741 (1978).

KAPPY, J\I., SELLINGER, S., and RAIZADA, M., Insulin binding in four regions of the developing rat brain. J. Neurochem. 42,198-203 (1984).

RAIZADA, M. K., YANG, J. W., and FE.LLOWS, R. E., Binding of 125I·Insulin to specific receptors and stimulation of nucleotide incorporation in cells cultured from rat brain. Brain Res. 200, 389-395 (1980).

SONNE, 0., and GLIEMANN, J., The mechanism of receptor· mediated degradation of insulin in isolated rat adipocytes. Indirect evidence for a nonlysosomal pathway. Molec. Cell. Endocrinol. 31,315-320 (1983).

"\VACHSMUTH, E. B., and STOYE, J. P., The differentiation of proximal and distal tubules in the male rat kidney: The appearance of aldolase isoenzymes, aminopeptidase, and alkaline phos­phatase during ontogeny. Histochemistry 47, 315-337 (1976).

YANG, J. W., RAIZADA, M. K., and FELLOWS, R. E., Effects of insulin on cultured rat brain cells. Stimulation of ornithine decarboxylase activity. J. Neurochem. 36, 1050-1056 (1981).

Authors' address: Dr. rer. nat. M. REISER, Institut fur Anatomie, Medizinische Akademie Magdeburg, Leipziger Str. 44, Magdeburg, DDR • 3090.