ALTERATION OF LACTASE AND AMINOPEPTIDASE N EXPRESSION IN PORCINE ENTEROCYTES BY ENERGY INTAKE AND ENVIRONMENT

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1 Quarterly Journal of Experimental Physiology (1986) 71, Printed in Great Britain ALTERATION OF LACTASE AND AMINOPEPTIDASE N EXPRESSION IN PORCINE ENTEROCYTES BY ENERGY INTAKE AND ENVIRONMENT M. J. DAUNCEY AND D. L. INGRAM A.F.R.C. Institute of Animal Physiology, Babraham, Cambridge CB2 4AT, England (RECEIVED FOR PUBLICATION 15 AUGUST 1985) SUMMARY The influence of nutrition, environmental temperature and time of feeding on lactase and aminopeptidase N activities in enterocytes of young pigs has been investigated. Animals were kept in the cold (10 C) or warm (35 C) and given either a high or low energy intake. In animals from the cold, lactase activity as determined by scanning microdensitometry was significantly lower at 4 h after a meal than it was in animals from the warm. At 6 h, the activities were similar in all groups, while at h after feeding, lactase activity was much greater in those kept at 10 C than in those at 35 'C. Level of energy intake had no statistically significant effect and no differences were found with respect to aminopeptidase N. The possibility that these differences in lactase activity are related to thyroid hormone metabolism and energy supply in relation to demand is discussed. INTRODUCTION Intestinal lactase in many mammalian species reaches a peak soon after birth and declines to low levels in the adult (Johnson, 1981). Controversy still exists as to whether this change in activity is regulated by the concentration of lactose in the diet. It is possible that the age-related appearance and disappearance of lactase activity is genetically controlled but that it may be stimulated by environmental factors (Kretchmer, 1981). One idea is that the energy supply in relation to metabolic demand is of importance. This has been investigated in the present study in young pigs which have been kept in a warm or cold environment on a high or low level of energy intake. The advantage of such a model is that it allows comparisons between animals on the same energy intake but with different metabolic demands made by the environment (Dauncey, Ingram, Walters & Legge, 1983). A separate possibility is that feeding itself influences the activity of some digestive enzymes. In adult rats, maltase and sucrase follow an endogenous circadian rhythm in which feeding acts as a Zeitgeber (Saito, Murakami, Nishida, Fujisawa & Suda, 1975; Stevenson & Fierstein, 1976; Saito, Sato & Suda, 1978, Saito, Kato & Suda, 1980). Enzyme activity increases before the start of a meal and reaches a peak by the end of feeding, but the rhythm persists even when the animals are deprived of food for 24 h. By contrast, lactase activity in the rat increases 24 h after withdrawal of food and appears to reach a maximum after a further 24 h (Raul, Noriega, Doffoel, Grenier & Haffen, 1982). Moreover, this increase is a consequence of the synthesis of lactase which is regulated at the translational level (Nsi-Emvo & Raul, 1984). However, aminopeptidase N is not affected by starvation over a period of 48 h. The second aim of the present investigation was therefore to determine the influence of time of feeding on lactase and aminopeptidase N activities.

2 444 M. J. DAUNCEY AND D. L. INGRAM METHODS Animals Forty-eight pigs of the Large White breed were used. The advantage of this species is that young pigs continue to grow under a very much wider range of environmental temperature and energy intake than do small rodents. Animals were taken in groups of four litter-mates at 14 d of age and housed in separate pens at a thermally neutral temperature of 30 'C. During the following two weeks, for two animals the temperature was gradually reduced to 10 'C and for the other two it was increased to 35 'C. At each temperature, one of the pigs was given a high energy intake and the other a low intake of the same commercial feed (protein 23%, gross energy 19 kj/g). There were thus four treatment groups: 35 high, 35 low, 10 high and 10 low. The amount of food given was increased as the animals grew but the ratio of high:low was always 2:1 (Dauncey et al. 1983). The food was provided as one meal/d and when the animals were killed at d of age, this amounted to 700 and 350 g for high and low respectively. Experimental techniques Animals were investigated at 4, 6 or h after feeding. In the cold, the daily ration of food was eaten rapidly; but in the warm the duration of the meal could be as much as 4 h, especially for those on the high intake, but all the food was eaten. Therefore, to standardize the time since feeding more precisely, only one-third of the ration was given on the day of examination for the 4 and 6 h periods of investigation and all the food was eaten within 30 min. Animals were pre-medicated with ketamine and killed by intracardiac injection of sodium barbiturate. The small intestine was immediately dissected free and a sample removed from the mid-point and washed with saline. A small portion was then frozen rapidly in isopentane over liquid nitrogen and stored at -20 'C. Lactase activity was examined by the method developed by Lojda, Slab', Kraml & Kolinska (I9'/3). Frozen sections, 10 #tm thick, were incubated at 37 'C for 10 min at ph 6 0 in the presence of 1 09 mrv; 4-Cl-5-Br-3-indolyl-/J-D-fucopyranoside. Aminopeptidase N was determined in 10,um serial sections incubated for 2 min at 37 'C in a medium containing 0-7 mm L-alanine-4-methoxy-fl naphthylamide at ph 6 5 as described by Nachlas, Crawford & Seiligman (1957). The intensities of the enzyme reaction products were measured by scanning microdensitometry at wave-lengths of 680 and 510 nm for lactase and aminopeptidase N, respectively. Incubation times were chosen to ensure that initial rates of hydrolysis were being measured. The scanning spot was moved over the enterocytes along the edge of the villus to provide a series of measurements corresponding to different distances from the crypt-villus junction. Three villi were scanned for each animal to obtain a mean value at each point along the villus which was then used to calculate the over-all mean for each treatment. Analysis of results The increase in enzyme activity as the enterocytes moved from the crypt towards the tip of the villus was evaluated by fitting the results obtained from the scanning densitometer for the rising phase of the curve according to the equation: y =A + 1 +e-b(x-m) where y was the density reading, x was the distance from the crypt-villus junction, A was fixed at 0 since it was assumed that the enzyme activity was initially zero, C was the calculated maximum activity, M was the distance from the crypt-villus junction when the enzyme activity was 50% of the maximum and B was proportional to the rate at which the activity of the enzyme increased. The fitting of the curve was carried out by a computer maximum-likelihood program (Ross, 1980). The significance of differences in the estimates of C, M and B were determined by the analysis of variance. RESULTS Lactase activity in enterocytes at increasing distances along the villus are given in Fig. 1 for each of the treatment groups and times after feeding. In all cases, enzyme activity rose sharply with increasing distance from the crypt-villus junction, reached a plateau at about

3 ENVIRONMENTAL EFFECTS ON LACTASE ACTIVITY 445 A high 35 low 10 high 10 low 4 - B 16 0 _...I.L4 I L l.l.l.i = high 35 low 10 high 10 low C 35 high 35 low 10 high 10 low Distance from crypt-villus junction (mm) Fig. 1. Mean values of lactase activity in enterocytes, in arbitrary absorbance units obtained by scanning microdensitometry. Values are plotted along the length of the villus from the junction with the crypt (left of curve) to its tip (right of curve). Results are for litter-mates kept at 35 or 10 C on a high or low level of energy intake; n = 4 for each group. Measurements were made at three different times in relation to the last meal: A, 4 h after feeding; B, 6 h after feeding; and C, h after feeding. half-way along the villus and declined precipitously towards the tip of the villus. The over-all results were strikingly different for the groups at the two environmental temperatures. Those from the cold showed a marked increase in lactase activity with increasing time since the last meal, and there was a tendency for this increase to be greatest in the animals on the low food intake. However, for animals kept in the warm, the results for the two levels of intake and three times after feeding showed little variation. By contrast with the results for lactase, aminopeptidase N activity was already high at the crypt-villusdjunction. Fig. 2 shows that the percentage increase in activity at the mid-point of the villus was much less than for lactase. Only at 4 h after feeding was there a slight

4 446 M. J. DAUNCEY AND D. L. INGRAM A high 35 low 10 high IO low , B 0 L : high 35 low 10 high 10 low 20 v 12 o E 1. 0 lll ll l W C high 35 low 10 high 10 low.0 Z O Distance from crypt-villus junction (mm) Fig. 2. Mean values of aminopeptidase N activity in enterocytes, in arbitrary absorbance units obtained by scanning microdensitometry. Values are plotted along the length of the villus from the junction with the crypt (left of curve) to its tip (right of curve). Results are for litter-mates kept at 35 or 10 'C on a high or low level of energy intake; n = 4 for each group. Measurements were made at three different times in relation to the last meal: A, 4 h after feeding; B, 6 h after feeding; and C, h after feeding. tendency for there to be lower enzyme activity in those from the cold but the variation between animals was high and the difference was not significant. Over-all, aminopeptidase N activity was not influenced by energy intake, environmental temperature or time since the last meal. The results for this enzyme thus acted as controls for the differences noted in lactase activity. More detailed analysis of the findings for lactase was carried out by fitting a logistic curve to the rising phase of the results shown in Fig. 1. Estimates of the maximum enzyme activity, C, are given in Table 1 for both the combined and separate effects of diet and temperature. No significant differences for the values of M or B were found in any of the groups. In animals examined only 4 h after feeding (Fig. 1 A), those at 10 'C were found to have

5 ENVIRONMENTAL EFFECTS ON LACTASE ACTIVITY 447 Table 1. Maximum activity (C) of lactase in enterocytes of young pigs kept at 35 or 10 C on a high or low energy intake and examined at various times after feeding. Least-squares mean values from the analysis of variance are given in arbitrary absorbance units Time after Temperature x energy intake Energy intake Temperature ( C) feeding (h) 35 high 35 low 10 high 10 low High Low P values from the analysis of variance Living temperature Energy intake 4 <001 N.s. 6 N.s. N.s <001 N.s. N.s., not significant. maximal lactase activities significantly lower than those at 35 C (Table 1). Indeed, in one litter at 10 C the enzyme activities were close to zero. It must be emphasized however that the difference related to temperature was significant even when this litter was excluded from the analysis. The values for lactase activity 6 h after feeding were similar in animals at 10 and 35 C and on both diets (Fig. 1 B) and the estimates of C were not statistically significant. In the animals living in the cold, maximal lactase activity at h after feeding was significantly greater than after 6 h (P < 0-01). Fig. 1 C shows that activities at h after feeding were much greater in animals at 10 than at 35 'C. It also shows that within each temperature, the activities tended to be greater in animals on the low than the high energy intake. Estimates of C were significantly greater in the cold than the warm (Table 1) whereas there was no significant effect of level of energy intake nor any interaction between temperature and intake. DISCUSSION The present results show that environmental temperature can have a striking influence on lactase activity in enterocytes of the young pig. Animals which have been living in the cold show a much greater change in enzyme activity in relation to the time of feeding than do those in the warm. Thus, at 4 h after feeding, enzyme activity is significantly less in animals which have been living at 10 compared with 35 'C; whereas h after the last meal, enzyme activity is significantly greater at 10 than at 35 'C. By contrast, level of energy intake had no significant effect. The animals in this investigation received the same two levels of food intake at both environmental temperatures. However, since for any given intake they grew more rapidly at 35 than 10 'C, the food intake as a percentage of body weight was actually greater for those in the cold. Therefore, the greater lactase activity at h after feeding in those at 10 compared with 35 'C cannot be attributed to those in the cold receiving less food, either in absolute terms or in relation to body weight. The animals living in the warm tended

6 448 M. J. DAUNCEY AND D. L. INGRAM to eat more slowly than those in the cold and hence the time since feeding was closer to 20 h for those at 35 C and nearer 24 h for those at 10 'C. However, it seems unlikely that lactase activity would have increased significantly in those at 35 'C if they had been kept a further 4 h before examination. A more probable explanation for the greater circadian variation in lactase activity seen in animals at 10 'C is that it is related to the energy intake in relation to the metabolic demands made on the animal. Thus, for a given level of energy intake pigs living at 10 'C have a resting metabolic rate (r.m.r.) which is almost double that of their litter-mates at 35 'C (Macari, Ingram & Dauncey, 1983). The greater r.m.r. in the cold is associated with hormonal differences and it is already known that the regulation of lactase is influenced by thyroid hormones. In the adult rat, both thyroidectomy and starvation result in increased lactase activity, while the administration of thyroxine (T4) prevents this increase in starved animals (Raul, Noriega, Nsi-Emvo, Doffoel & Grenier, 1983). In pigs acclimated to 10 or 35 'C on a high or low level ofenergy intake, the rates at which T4 and 3,5,3'-triiodothyronine (T3) are metabolized increase both in the cold and on a high intake (Macari, Dauncey, Ramsden & Ingram, 1983). Moreover, Dauncey, Ramsden, Kapadi, Macari & Ingram (1983) found that the plasma concentration of T3 increases within 1-2 h of eating a meal. The possibility is therefore that the considerable fall in lactase activity 4 h after feeding in pigs at 10 'C depends at least in part on the action of thyroid hormones. However, if lactase is particularly sensitive to the action of thyroid hormones it might have been expected that a clear-cut difference related to the level of energy intake would also have been found. In a study of the influence of T4 on lactase expression in adult rats, Hewitt & Smith (1984) found that although the hormone caused a decrease in lactase after 48 h, it resulted in an increase after only 8 h. This increase in enzyme activity was particularly marked in enterocytes which had already moved up the villus and were therefore older. Young pigs kept in the cold maintain enterocytes on the villus for about 5000 more time than their litter-mates in the warm (Dauncey, Ingram, James & Smith, 1983). It might therefore be expected that they would also be more sensitive to thyroid hormones. It is thus possible that although lactase activity is lost rapidly immediately after a meal from animals in the cold, it also recovers rapidly because the enterocytes are older than those from animals in the warm. By contrast with the findings for lactase, the activity of aminopeptidase N showed no significant variation in relation to any of the parameters studied. Therefore, whatever the explanation for the differences in lactase activity, it is unlikely to be related to some general effect on digestive enzymes. Nevertheless, if the positions of lactase and aminopeptidase N on the brush border of the enterocytes differ, lumenal degradation by pancreatic or bacterial proteases (Alpers, 1983) and physical damage by food could become important. It therefore remains to be established whether environmental and hormonal factors can influence the topographical situation of the brush border enzymes. We thank Mr I. King for his skilled preparation of the histological specimens; Mr D. Brown, A.F.R.C. Statistics Group, for his help and advice; and Mr K. F. Legge for management of the animals. REFERENCES ALPERS, D. H. (1983). Protein synthesis and turnover in the mammalian intestine. In Protein Metabolism and Nutrition, ed. ARNAL, M., PION, R. & BONIN, D., pp Paris: Les Colloques de 'I.N.R.A., no. 16.

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