Biochimica et Biophysica Acta 924 (1987) 257-259 257 Elsevier

BBA 20201 BBA Report

Substrate specificity in the stimulation of intestinal ornithine decarboxylase activity by refeeding after starvation

A.R. Baer, C.I. Cheeseman and A.B.R. Thomson Departments of Physiology and Medicine, University of Alberta, Edmonton, Alberta (Canada)

(Received 26 August 1986) (Revised manuscript received 5 January 1987)

Key words: Substrate specificity; Ornithine decarboxylase; Refeeding; (Rat intestine)

Jejunal loops in 3-day-fasted rats were pedused with hexoses and amino acids to test for their ability to stimulate intestinal ornithine decarboxylase activity. Intraluminai L- and D-glucose, galactose and 3 -0 - methylglucose were potent stimulants, while D-fructose and L-leucine were not. Intravenously infused D-glucose was also without effect. Induction of oruithine decarboxylase therefore appears to involve a receptor-mediated event which is probably located at the luminal cell sudace.

Several adaptive changes occur in the intestine during starvation, including mucosal hypoplasia and a reduction in the rate of cell proliferation [1,2]. The uptake rate of actively transported sub- strates such as glucose is also decreased [3]. Dep- rivation of luminal nutrition alone appears to account for these atrophic changes, as similar decreases are observed during parenteral nutrition [4]. Refeeding induces a rapid return to the pre- starvation state [1,5], and coincidentally, there is a large increase in the activity of the enzyme ornithine decarboxylase in the intestine, which peaks 3-5 h after the reintroduction of food [6]. Ornithine decarboxylase synthesizes the first of a series of compounds collectively known as poly- amines, which are known to play a role in the regulation of DNA synthesis [7]. It seems likely, therefore, that ornithine decarboxylase is an im- portant mediator in the return of both normal intestinal function and morphology following refeeding.

Correspondence: C.I. Cheeseman, Department of Physiology, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.

The exact stimulus for ornithine decarboxylase induction after refeeding is uncertain. Recently, however, Minami et al. have reported that perfus- ing the intestinal lumen of the rat with amino acids such as glycine and alanine stimulated mucosal ornithine decarboxylase activity, whereas other amino acids such as arginine or lysine did not [8]. Further, intraperitoneal injection of glycine or alanine failed to induce activity of the enzyme. This suggests that intestinal ornithine decarboxy- lase activity depends on a luminal stimulus and is induced only by specific substrates. The present study was undertaken to determine whether carbohydrates can also act as a stimulus for induc- tion of ornithine decarboxylase activity, and whether the manner in which the substrate enters the cell in any way determines whether the induc- tion occurs.

Male Sprague-Dawley rats weighing approxi- mately 250-300 g were starved for 3 days before a silicon cannula was implanted under pentobar- bital anesthesia in the mid-jejunum for in vivo substrate infusions. A ligature placed immediately proximal to the cannula insertion prevented reflux of intestinal contents into the stomach. In one

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group, a cannula was implanted in the right jugu- lar vein for parenteral substrate infusion. Animals were then infused with one of two concentrations of substrate for 4 h at a rate of 5 m l / h using a Sage infusion pump.

One set of animals was infused with a 555 mM (10% w / v ) solution of either D-glucose, D-galac- tose, or D-fructose. The parenteral group received D-glucose. The high substrate concentration used in this set allowed the study of the effects of hyperglycemia in the blood compartment and avoided negative results due to subthreshold sub- strate concentrations. Another set received a 100 mM solution of o-glucose or L-glucose (with or without 2 mM phloridzin), L-leucine, or 3-O-meth- ylglucose. In this group, NaC1 was added to main- tain isosmolarity. Controls were also starved for 3 days, and were implanted with a jejunal cannula but were not infused. No D-glucose could be de- tected in the L-glucose solution with a Beckman glucose analyzer. Ornithine decarboxylase activity was measured in mucosal scrapings using the method of Pegg and McGill [9]. Results were expressed per mg D N A according the method of Holt et al. [10]. Plasma immunoreactive insulin levels were determined using a dextran-coated charcoal separation technique [11] in fasted animals which were infused with a 1.1 M (20% w / v ) glucose solution at a rate of 3 ml /h .

555 mM D-glucose administered luminally elicited a substantial increase in ornithine de- carboxylase activity compared to fasted controls, whereas the same solution delivered intravenously failed to stimulate enzyme activity (Table I). D- galactose stimulated ornithine decarboxylase ac- tivity to the same degree as D-glucose, whereas no difference from controls was observed when D-fructose was infused.

At the lower substrate concentration (Table II), D-glucose was still able to stimulate ornithine de- carboxylase activity, although to a significantly lower degree than with the 555 mM solution ( P < 0.05). Enzyme activity was also elevated by 3-0- methylglucose and by L-glucose, but not by L-leucine. Enzyme induction by either L- or D-glu- cose was abolished by simultaneously infusing 2 mM phloridzin (a specific inhibitor of Na+-depen - dent hexose transport across the brush-border membrane). Adjacent intestinal segments which

T A B L E 1

S T I M U L A T I O N O F I N T E S T I N A L O R N I T H I N E D E -

C A R B O X Y L A S E A C T I V I T Y BY 555 m M S U B S T R A T E

O r n i t h i n e d e c a r b o x y l a s e act iv i ty

( p m o l / h per m g D N A )

C o n t r o l 98.7 + 32.3

D - G l u c o s e 1041.1 -+ 173.2 ~

D-Glucose , i.v. 141.0-+ 41.3

D - F r u c t o s e 36.4-+ 12.9

D - G a l a c t o s e 790.3 -+ 169.6

~ P < 0.005 vs. cont ro l .

n = 5 - 7 pe r subs t ra te .

were not infused showed no evidence of ornithine decarboxylase induction.

These results clearly indicate that the increase in ornithine decarboxylase activity that coincides with refeeding after starvation is dependent on the nature of the substrate administered. This stimula- tion of intestinal ornithine decarboxylase activity by hexoses shares most of the features previously reported for stimulation mediated by amino acid feeding [8], including induction by both active and passively transported substrates, a dependence on luminal delivery of the substrate, and the finding that substrate metabolism is not required.

D-Galactose and 3-O-methylglucose both use the glucose cartier but are not metabolized by the enterocyte [12,13], yet both acted as a potent stimulus. Transport across the brush-border mem- brane via sodium-dependent mechanisms does not

T A B L E II

S T I M U L A T I O N O F I N T E S T I N A L O R N I T H I N E D E -

C A R B O X Y L A S E A C T I V I T Y BY 100 m M S U B S T R A T E

O r n i t h i n e d e c a r b o x y l a s e ac t iv i ty

( p m o l / h per m g D N A )

C o n t r o l 98.7 _+ 32.3 D-Glucose 361.6_+ 67.0 ~'

D - G l u c o s e + p h l o r i d z i n 2 2 . 4 + 5.6 a

L -Glucose 765.0 + 237.0 b

L - G l u c o s e + p h l o r i d z i n 12.1 _+ 5.1 ~

t - L e u c i n e 69.9--+ 20.8

3 - O - M e t h y l g l u c o s e 631.5 _+ 201.8 b

a p < . 0 5 , b p < 0.1 vs. con t ro l .

n = 5 - 7 per subs t ra te .

TABLE III

ELEVATION OF PLASMA INSULIN LEVELS BY VASCU- LAR GLUCOSE INFUSION

Plasma insulin levels (ng/ml) are given as mean values _+ S.E.

Plasma insulin

Control 0.17 + 0.06 10 min 3.67 +_ 0.78 20 min 5.29 + 0.99 40 min 7.26 _+ 1.40 60 min 7.72 + 1.18

appear to be the common factor involved in the stimulation of the enzyme, however. While fruc- tose, whose transport is sodium-independent, had no effect, neither did L-leucine, which does require sodium co-transport. In addition, L-glucose, which has only a very low affinity for the D-glucose transport system [14], was a potent stimulus, out of proportion to its low rate of uptake. Vascular infusion of glucose had no effect on ornithine decarboxylase levels but caused a very significant elevation of plasma insulin levels (Table III). This rules out a role for insulin in the response, or a serosal site for the action of this stimulus. Previ- ous work demonstrating the stimulation of intesti- nal ornithine decarboxylase activity by high-dose insulin [6] is therefore probably a consequence of hypoglycemia leading to catecholamine release, the latter being a known inducer of ornithine decarboxylase activity [15]. As phloridzin cannot enter the enterocyte [16], the ability of this inhibi- tor to block the action of both D- and L-glucose suggests the involvement of a receptor probably on the surface of the brush-border membrane. At present it is not possible to state whether one receptor binds both the hexoses and the amino acids but it is clear that the receptor(s) is not stereospecific.

The results obtained here are consistent with previous work with parenterally fed rats in which luminal nitrition was required to maintain optimal

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intestinal function [4] and parameters such as villus height. Previous investigators were puzzled by the fact that intestinal function could also be maintained by compounds such as 3-O-methylglu- cose which have no nutritional value [17]. The present study would appear to indicate that if ornithine decarboxylase activity is connected with intestinal function and morphology, maintenance of these parameters may depend not on the nutri- tive value of the luminal contents, but rather on whether they have the proper structural configura- tion to bind a luminal receptor.

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