0013-7227/78/1024-1118$02.00/0 Endocrinology Copyright © 1978 by The Endocrine Society

Vol. 102, No. 4 Printed in U.S.A.

Effect of Alloxan-Induced Diabetes on Intestinal Peptidases in the Rat* CONSTANTINE ARVANITAKIS,t JOHN FOLSCROFT, AND GRETCHEN STITT Department of Medicine, Division of Gastroenterology, Kansas University School of Medicine, Kansas City, Kansas 66103 ABSTRACT. Intestinal transport of amino acids, similar to sugar absorption, is enhanced in experimental diabetes. Because peptidases play a significant role in peptide digestion, we examined the effect of diabetes on intestinal peptidases. Leucyl-naphthylamidase and leucyl-glycine hydrolase (brush border peptidases) and prolyl-glycine hydrolase (cytosol peptidase) were assayed in the brush border and cytosol fraction in diabetic rats 7 days after alloxan administration. Mucosal weight, protein concentration, and total and specific activity of

E

XPERIMENTAL diabetes induced by alloxan or streptozotocin is associated with enhanced intestinal transport of sugars (1-3) and amino acids (2,4). In addition, brush border enzymes, namely disaccharidases, are increased in chemically induced diabetes (5, 6). Thus, it appears that both the absorptive and digestive function of the small intestinal mucosa is altered in diabetes. Inasmuch as amino acid transport is increased in diabetes and intestinal peptidases are important enzymes in the digestion and absorption of peptides and amino acids, it was of interest to examine the effect of experimental diabetes on intestinal peptidase activity. Because of the different subcellular localization of peptidases, we studied the effect of diabetes on representative membrane-bound and cytoplasmic peptidases in the rat small intestine. Materials and Methods Rats Male Holtzman rats weighing 250-310 g were fed commercial laboratory chow ad libitum and alReceived January 27, 1977. * This work was supported by grants from the Kansas University Endowment Association. f To whom all correspondence and requests for reprints should be addressed at: Department of Medicine, Kansas University Medical Center, Rainbow Boulevard at 39th Street, Kansas City, Kansas 66103.

leucyl-naphthylamidase and leucyl-glycine hydrolase were significantly increased in diabetes in the brush border but not in cytosol fraction. By contrast, prolylglycine hydrolase was not affected in cytosol fraction or brush border. These data indicate that brush border peptidases are increased in experimental diabetes. This adaptive response of the small intestinal mucosa is similar to disaccharidase elevation and alteration in the intestinal absorptive function which occurs in experimental diabetes. (Endocrinology 102: 1118, 1978)

lowed free access to water. Purina chow diet consisted of 23% protein, 4.5% fat, 6% fiber, and 50% nitrogen-free extract. Rats were randomly allocated to receive alloxan or normal saline. After an overnight fast, a group of rats was injected via the tail vein with alloxan monohydrate (40 mg/kg). The control group was injected with the same volume of normal saline. The animals were housed in metabolic cages and 24-h urine was collected and tested for glucose (Labstix, Ames Co., Inc., Elkhart, IN). Daily food consumption was recorded in both groups of animals. Criteria for experimental diabetes included 1) persistent glucosuria, 2) hyperglycemia (blood glucose > 250 mg/dl), and 3) weight loss. Brush border preparations Rats were sacrificed on the 7th day after alloxan administration. The entire small intestine, from the ligament of Treitz to the ileocecal valve, was removed, flushed with cold 0.9% NaCl, and divided into two segments of equal length, proximal and distal. The same proportion of the entire small intestine was removed from the control and diabetic rats. The intestinal segments were then everted on a glass rod and mucosal scrapings were obtained with a glass slide. Mucosa from each animal in each group was weighed and homogenized in cold water in a Waring Blender for 15 sec. After centrifugation at 3000 X g at 4 C for 15 min, the pellet was washed three times by suspension and centrifugation in 5 mM EDTA adjusted to pH 7.4 with NaOH. Brush borders from control and diabetic rats

1118

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 13:06 For personal use only. No other uses without permission. . All rights reserved.

1119

INTESTINAL PEPTIDASES IN DIABETES were prepared according to the method of Forstner, Sabesin, and Isselbacher (7). The preparations were free of significant contamination, as determined by the specific activity of prolyl-glycine and prolylglycyl-glycine hydrolases, which are cytosolic enzymes (8) and by the enhancement of specific sucrase activity (9), a marker of brush border enzymes. Sucrase specific activity was approximately 20-fold higher in the brush border preparation than in mucosal homogenates. Negligible sucrase activity was detected in the cytosol fraction ( fc

3

CD g : ,-. p

EL-5 2 ^ 3 »

a 3

3

3

^ 3'

o 3 P jj

?" 3

e5

S 2.

co O

* e li co 5 ' 8 I?

n =

* i-1 5' 3 O*

Qj ^S

? r^

P

to o-

® ? 3

I iiiff O

o

' ? ??

2 S

» §-. ??

el- O

d

CO t-s (0

5- c

s gr §C"cr° cr p' p o o e ^ §^ §8 8o 5'

^ aq

» g' 3 ^

§"1 -a-* Q. £J

p

w

2 . Co CTQ C O

^

fD fD P CL

co S , 3 ; oO OQ 2. p" § „

P

•c

i— • fD

s1-

H ?r cr

» - CO to

fTg Pas.. o ro

|I

«5* H B5 ff

o

595 ± 96