Regulation of Rat Proximal Intestinal Glycolytic Enzyme Activity by Ileal Perfusion With Glucose

GASTROENTEKOI.OGV 71 :295-298. 1976 Copyright «J 1976 by The William, & Wilkins Co.

Vol. 71 . No 2

PrintM jn U.S .A .

REGULATION OF RAT PROXIMAL INTESTINAL GLYCOLYTIC ENZYME ACTIVITY BY ILEAL PERFUSION WITH GLUCOSE JULIO ESPINOZA,

M.D.,

SUSANNE BENNE'IT CLARK,

NORTON S . ROSENSWEIG,

Ph.D.,

ANN HRITZ,

B.S.,

AND

M .D.

Division of Gastroenterology. Medical Service. St . Luke's Hospital Center. New York, New York 10025

Specific activities of the glycolytic enzymes fructose-l -phosphate aldolase, fructose-l,6-diphosphate aldolase, and pyruvate kinase, are higher in rat duodenum and jejunum than in ileum. Whether this gradient reflects the failure of't1ietary sugars to reach the ileum in high concentrations is unknown. Rats were first fed a carbohydrate-free diet for 3 days, which virtually removed the proximal-distal gradient of enzyme specific activities. Twenty percent glucose was then perfused directly into either the duodenum or the ileum for 3 days. Both proximal and distal glucose perfusion restored the normal gradient of all three enzymes. Ileal pyruvate kinase was also increased by ileal glucose perfusion, but ileal aldolases were no higher after distal perfusion than after duodenal perfusion . The low ileal aldolase levels normally found in fed rats therefore are an intrinsic property of distal intestine and are not due to failure of dietary sugar to reach this portion of the gut. Furthermore, adaptation of duodenal and jejunal glycolytic enzymes to ileal glucose perfusion suggests a humoral and/or neural mechanism rather than a direct local luminal effect of the sugar itself. Previous studies have demonstrated that rat jejunal glycolytic enzyme activities adapt to dietary sugars. H High carbohydrate diets produced higher enzyme activities than did isocaloric carbohydrate-free or fasting regimens. More recent studies have shown that the adaptive response occurs along the entire small intestine. 4 However, the responses of the duodenal and jejunal glycolytic enzyme activities are much greater than those of the ileum . In addition, there appears to be a proximal-distal gradient of glycolytic enzyme activi· ties; for example, fructokinase activity is highest in the duodenum and jejunum and lowest in the ileum. 5 •• From none of these studies, however, is it possible to determine whether the higher activities in the proximal intestine reflect the normally higher concentration of dietary substrate in the proximal lumen, or whether the gradient is an intrinsic characteristic of small bowel enzyme activity. It is also not clear whether the adaptive response of mucosal glycolytic enzyme activities to ingested carbohydrate is due to local intraluminal fac tors, or whether it reflects a message transmitted ex traluminally, possibly via humoral and/or neural routes. Accordingly, the present study was undertaken to compare the effects of ileal glucose perfusion with those Received November 25, 1975. Accepted February 9, 1976. This work was supported by Grants AM 13436. AM 0&499, AM 14122. and AM 17685 from the National Institutes of Health, and by International Postdoctoral Research Fellowship 1 FOI; TW-1750. Dr. Espinoza's work was supported in part by a grant from the Overseas Maritime Corporat ion . Dr. Espinoza's present address is: Departmento de Nutricion y Tecnolollia de 106 Alimentos. U niversidad de Chile Sede, Santiago Sur. Santiago. Chile.

of an indentical duodenal glucose perfusion on rat small intestinal glycolytic enzyme activities.

Methods Thirty-six male Wistar rats ages 6 to 8 weeks were minimally restrained in Bollman-type restraint cages (day 0). All were allowed water and a carbohydrate-free, high casein diet I ad libitum throughout the 8 days of the study. The animals were allowed to become accustomed to the restraint for 3 days. On day 3, they were anesthetized with ether, a small midline incision was made below the xyphoid cartilage, and a polyethylene cannula (PE 10) was surgically implanted into either the first portion of the duodenum (three proximal groups; 18 animals) or the middle of the small intestine (three distal groups; 18 animals) through a small incision in the wall of the intestine. The cannulas were held in place by purse string sutures and were exteriorized through stab wounds in the abdomen. The animals were then returned to the restraint cages for a further 2 days before the test infusions were begun . Beginn ing on day 5, 6 proximally perfused and 6 distally perfused animals in each group received either 20% glucose or 0.85% NaCI for 3 days. These solutions were administered on an intermittent schedule, viz, 6-hr infusion periods at 4.58 ml per hr alternated with 6-hr periods of no infusion . All animals were killed on day 8 immediately after the last infusion period . Two sham control groups of 6 animals each, provided with either proximal or distal cannulas, were restrained but not infused for 8 days. The small intestine was quickly removed and divided into six segments of approximately equal length without washing the lumen-duodenum (D), three segments of jejunum (Jl, J2, J3) and two segments of ileum (11, 12). The mucosa of each segment was scraped immediately, weighed, and kept frozen at -80°C until glycolytic enzyme assays were performed within 24 to 48 hr. Preliminary experiments estab· lished that no significant loss of activity occurred when mucosa was frozen for this period.

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Enzyme determinations were carried out essentially accord- enzyme specific activities along the length of the small ing to Stifel et al. 1. • Briefly, scraped mucosa was homogenize
Results In agreement with previous data,' rats fed a carbohydrate-free diet (saline-infused and sham controls) showed a marked decrease in the normal gradient of

proximal enzyme increases. Aldolase activities were the

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FIG. 1. Regional mucosal F-I-P specific activities after luminal perfusion of 20% glucose for 3 days. The intestine was divided into six approximately equal length segments: D (duodenum); JI, J2, J3 (jejunum); and ll, 12 (ileum). Specific activities are given in nanomoles of product formed per minute per milligram of mucosal protein; mean ± SEM. e-e, Proximal glucose perfusion; e-----e, distal glucose perfusion; A-A, proximal saline perfusion; A-----A, distal I8line perfusion; 0--0, proximal sham; 0·----0, distal sham.

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FIG. 2. Regional mucosal F-l,6-P specific activities after luminal perfusion of 20% glucose for 3 days. e--e, Proximal glucose perfusion; e-----e, distal glucose perfusion; A-A, proximal saline perfusion; & ... --&, distal saline perfusion; 0--0, proximal sham; 0 .. ·--0, distal sham.

August 1976

JEJUNAL ENZYME ADAPTATION TO ILEAL GLUCOSE INFUSION

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FIG. 3. Regional mucosal PK specific activities after luminal perfusion of 20% glucose for 3 days . •--e, proximal. glucose perfusion; .-----., distal glucose perfusion; A--A, proximal saline perfusion; A-----A, distal saline perfusion; 0--0, proximal sham; 0--- --0, distal sham.

same as after proximal glucose infusion (figs . 1 and 2). However, PK in 12 was significantly increased after distal compared with proximal glucose administration (P < 0.02), tending to abolish the PK gradient (fig. 3). Thus, distal PK levels appeared to respond both to luminal and to nonluminal factors, in contrast to the aldolases, which showed no increase in distal specific activities after direct exposure to distal luminal glucose. In contrast to glucose, neither proximal nor distal saline infusion affected proximal enzyme activities. No differences in PK or aldolase levels in D, Jl, or J2 were noted in comparing sham controls with saline per~usi~n in general, nor proximal with distal saline perfusIOn In particular. However, J3, in which the cannula was located showed decreased aldolase levels (P < 0.01) when distal saline infusion was compared with the distal sham group. In addition, both J3 and 11 aldolases w~re decreased after distal compared with proximal sah~e infusion (P < 0.05). Thus, aldolases differed from PK In that they were decreased in distal intestine by local saline infusion. Discussion The present experiments demonstrate t.hat: aft~r 3 days on a carbohydrate-free diet, the adaptive mcreases in the mucosal glycolytic enzymes PK, F-I-P, and

297

F-l ,6-P to luminal perfusion of 20% glucose are the same whether the duodenum or the ileum is perfused. This suggests that the mechanism of the adaptive process is mainly mediated by nonluminal rather than by luminal factors. Most probably, adaptation to glucose involves humoral, or possibly even neural, mechanisms. In man, mucosal glycolytic enzyme levels and their adaptive responses to carbohydrate -containing diets may be modified by testosterone,IO insulin, \I and glucagon. \I In the rat, mucosal PK. but not F-l,6-P, was enhanced by oral testosterone or estradiol,12 although the same hormones given parenterally were ineffective. IS Other sex hormones also affected mucosal glycolytic enzyme levels ... Direct stimulation of mucosal glycolytic enzymes by elevated levels of glucose in mesenteric arterial blood may also occur. The intestinal pattern of glycolytic enzyme specific activities in rats maintained entirely by intravenous administration of high glucosecontaining solutions was found to be similar to the normal pattern (W. H. Heird and N. S . Rosensweig, unpublished data); and in analogous studies in man, I t jejunal PK, F-I-P, and F-l,6-P specific activities were significantly increased after intravenous 50% glucose infusion compared with a carbohydrate-free diet. However, glucose levels in mesenteric arteries were not determined in these experiments, and moreover, adaptive increases may nevertheless be mediated by hormonal mechanisms even in parenterally nourished animals. In this regard, the recent paper by Johnson and coworkers 16 is of interest. These authors found that, in rats, pentagastrin normalized the mucosal disaccharidase specific activities which had been suppressed by total parenteral nutrition for 10 days. Other studies in parenterally nourished man, U however, suggest that the adaptive responses of glycolytic enzymes are mediated differently from those of disaccharidases so that a hormone other than gastrin may be involved. The present experiments provide no insight as to whether only generating crypt cells are affected or whether mature villus mucosal cells respond to produce the increased enzyme concentrations. Other data, however, suggest that villus mucosal cells are affected directly. After oral sex hormones, near-maximal increases in jejunal mucosal PK were obtained within 8 hr in rats,l2 which is less than the generation time of rat crypt cells. 17 Similarly, in man, maximum increases in jejunal aldolases after sucrose feeding occurred after only 1 day. \I The present results obtained after duodenal perfusion of glucose differ slightly from those obtained after oral glucose administration.· The highest PK levels were now obtained in J2 instead of in Jl (fig. 1). Aldolase levels in J2 (figs . 2 and 3), although lower than in Jl, were also relatively higher than those obtained in J2 after oral glucose. Thus, the pattern of peak enzyme activity had shifted distally after duodenal perfusion compared with oral glucose. The reasons for the shift are unknown but may include such factors as stress resulting from the surgical interference and the prolonged restraint, the absence of other dietary components, or the rapid fluid infusion rate at high osmolarity. In general, however, the

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data from both types of experiments agree, because enzyme activities were in both cases much higher in proximal than in distal intestine. The present data also demonstrate that the distal adaptive response of PK differs from that of the aldolases. Thus, distal glucose perfusion produced an increase in distal PK activity above the levels induced by casein, thereby virtually abolishing the proximal-distal PK gradient found in normal and high carbohydrate-fed animals. It appears that PK can adapt to some degree to local perfusion of glucose. On the other hand, distal aldolase levels were decreased significantly by distal saline perfusion (figs. 2 and 3), although at present the mechanism for this effect remains obscure. The data of the present experiments have, however, clearly established that PK, F-I-P, and F -I,6-P activities in duodenal and jejunal mucosa respond equally after distal or proximal luminal glucose perfusion, that local factors may play some part in distal adaptation, and that the responses differ for the three enzymes studied. Most probably, the intrinsic gradient of glycolytic enzyme adaptation demonstrated by the present study is mediated by some, as yet undefined, humoral mechanism. REFERENCES

5. Anderson JW, Zakim 0: Enzyme activity of the intestine (abstr). J Clin Invest 48:3. 1969 6. Weiser MM. Quill H, Isselbacher KJ: Effects of diet on rat intestinal soluble hexokinase and fructokinase activities. Am J Physiol 321:844-849. 1971 7. Lowry OH, Rosebrough NJ, Farr AL, et al: Protein measurements with the Folin phenol reagent. J Bioi Chem 193:265-275, 1951 8. BucherT, Pfeiderer G: Methods in Enzymology. Vol. 1. New York. Academic. 1955, p 435--440 9. Spolter PO, Adelman RC, Weinhouse S: Distinctive properties of native and carboxypeptidase-treated aldolase of rabbit muscle and liver. J Bioi Chem 240:1327-1337, 1965 10. Lufkin EG, Stifel FB, Herman RH , et al: Effect of testosterone on jejunal pyruvate kinase activities in normal and hypogonadal males. J Clin Endocrinol Metab 34:586-591, 1972 11. Lufkin EG, Taunton 00. Stifel FB, et al: Effects of insulin, tolbutamide, and glucagon on activities of jejunal carbohydratemetabolizing enzymes in humans. Metabolism 24:923-928, 1975 12. Stifel FB, Herman RH. Rosensweig NS: Dietary regulation of glycolytic enzymes. Vlll. Dose and time response of rat jejunal enzymes to oral sex hormones. Biochim Biophys Acta 208:387-393, 1970 13. Stifel FB, Herman RH. Rosensweig NS: Dietary regulation of

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glycolytic enzymes. I. Adaptive changes in rat jejunum. Biochim Biophys Acta 170:221-227. 1968 2. Shakespeare p. Srivastava ZM. Huhscher G: Glucose metabolism in the mucosa of the small intestine. The effect of glucose on hexokinase activity. Biochem J 111:63-67, 1969 3. Stifel FB, Herman RH, Rosensweig NS: Dietary regulation of glycolytic enzymes. III. Adaptive changes in rat jejunal pyruvate kinase. phosphofructokinase, fructose diphosphatase and glycerol3-phosphate dehydrogenase . Biochim Biophys Acta \84:29-34,

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glycolytic enzymes. V. Lack of effect of intramuscularly administered sex steroid on male and female rat jejunum. Biochim Biophys Acta 208:368-373, 1970 Stifel FB, Herman RH, Rosensweig NS: Dietary regulation of glycolytic enzymes. IV. Differential hormonal effects in male and female rat jejunum. Biochim Biophys Acta 184:495-502. 1969 Greene HL, Stifel FB. Hagler L. et al: Comparison of the adaptive changes in disaccharidase, glycolytic enzyme and fructose diphosphatase activities after intravenous and oral glucose in normal men. Am J Clin Nutr 28:1122-1125, 1975 Johnson LR, Lichtenberger 1M. Copeland EM. et 01: Action of gastrin on gastrointestinal structure and function. Gastroenterology 68:1184-1192. 1975 Hanson WR, Osborne JW: Epithelial cell kinetics in the small intestine of rat 60 days after resection of 70 per cent of the ileum and jejunum. Gastroenterology 60:1087-1097. 1971 Rosensweig NS, Stifel FB, Zakim 0, et 81: Time response of human jejunal glycolytic enzymes to a high sucrose diet. Gastroenterology 57: 143-146. 1969