Effects of fasting on mucus glycoprotein biosynthesis in rat stomach

Comp. Biochem. Physiol. Vol. 82B, No. 2, pp. 207-210, 1985 Printed in Great Britain

0305-0491/85 $3.00 + 0.00 © 1985 Pergamon Press Ltd

EFFECTS OF F A S T I N G ON M U C U S G L Y C O P R O T E I N BIOSYNTHESIS IN RAT STOMACH S. OHARA a n d K. HOTTA* Department of Biochemistry, School of Medicine, Kitasato University, Sagamihara, 228, Japan (Received 25 February 1985) A b s t r a c t - - l . Biosynthetic activity of gastric mucus glycoprotein in rats after fasting for 24 and 72hr was

studied by the organ culture technique. 2. Fasting produced a slight reduction in gastric mucus glycoprotein biosynthesis in the corpus and antrum (about 70-90%0 of fed rats). 3. Sulfation of gastric mucus glycoprotein was restrained in the corpus (18% in control for 72hr).

INTRODUCTION Gastric mucus glycoprotein is the chief c o m p o n e n t of mucus gel which is considered to protect surface mucosal cells (Horowitz, 1977; Allen, 1981). Gastric mucus glycoprotein is a suit of armor, so to speak, against irritants a n d digestive enzymes (Takagaki a n d Hotta, 1979), a n d allows solid material to pass easily t h r o u g h the gut (Winet, 1976). Internal factors, i.e., HC1 a n d pepsin a n d external factors such as foodstuffs, influence secretion, a c c u m u l a t i o n a n d the biosynthesis of gastric mucus glycoprotein. The m e t a b olism of gastric m u c u s glycoprotein is correlated with ulceration. In a previous study, the a d m i n i s t r a t i o n of aspirin to rats was f o u n d to cause a decrease in gastric mucus glycoprotein content before macroscopinal changes in the mucosa, followed by the d e v e l o p m e n t of gastric ulcers (Azuumi et al., 1980). P a r k e a n d Symons d o c u m e n t e d the relation between various factors a n d the biosynthesis of gastric mucus glycoprotein: ulcerogenic drugs and stress inhibit the biosynthesis of gastric m u c u s glycoprotein, while anti-ulcer drugs ( p r o s t a g l a n d i n a n d carbenoxolone) increase its biosynthesis (Parke a n d Symons, 1979). Similarly, the metabolism of gastric m u c u s glycoprotein changes according to the transition o f physiological p h e n o m e n o n . Dekanski reported a reduction in the synthesis o f gastric glycoprotein in fasted rats (Dekanski et al., 1975). In o u r previous paper, we reported t h a t m u c u s glycoprotein accumulates in the corpus m u c o s a d u r i n g fasting and t h a t its acidity decreases ( O h a r a et al., 1984). O u r previous results a n d D e k a n s k i ' s data m a y be considered mutually confirmative, assuming t h a t fasting suppresses the secretion a n d / o r d e g r a d a t i o n of mucus. F o r clarification of this point, the effects of fasting on the biosynthetic rates of m u c u s glycoprotein were investigated in the present research as well as those o f fasting on the sulfation of mucus glycoprotein. MATERIALS AND METHODS

Male Wistar albino rats weighing 150-170g (Shizuoka Agricultural Corp. Assoc. Lab., Japan) were used through-

*To whom requests for reprints should be addressed.

out the course of the present experiments. The animals were made to fast for 24 or 72 hr, but always had free access to water. During these periods, they were housed in wire bottomed cages to prevent coprophagy. The control animals were fed ad libitum before the experiments. The rats were killed by exsanguination from the carotid artery, and their stomachs were excised and washed in Ca 2÷, Mg 2÷ free phosphate buffered saline. Corpus and antrum specimens were selected macroscopically and excised. To assess the biosynthesis of gastric mucus glycoprotein, the organ culture technique was used. The incorporation of [3H]glucosamine and [3SS]sulfate into gastric mucus glycoprotein was measured by the in vitro incorporation procedure as previously described (Ohara et al., 1983). Rat gastric corpus and antrum were sliced into approximately 2 mm squares. The tissue was immersed in 90% Eagle's minimum essential medium and 10% dialysed fetal calf serum, followed by incubation at 37°C in a humidified: environment of 5% CO 2 and 95% air for 5hr. D-[6-3H]Glucosamine (30 Ci/mmol: New England Nuclear) and [3SS]sulfate (carrier free: Amersham) were added to the medium at activities of 10 and 50 ,uCi/ml, respectively. After incubation, the tissue was harvested from the medium after being rinsed 3 times with 2 ml of Ca 2+, Mg z+ free phosphate buffered saline. The gastric mucus glycoprotein (Fr-I) was isolated from the tissue as described previously (Ohara et al., 1982, 1983). To isolate the gastric mucus glycoprotein from the medium, the medium was centrifuged at 3000 rpm for 15min. The supernatant was chromatographed with Bio-Gel A-1.5 m. Aliquots of the incubated tissue homogenate were taken for protein measurement by the modified Lowry's method (Wang and Smith, 1975). DEAE-Sepharose CL-6B column chromatography was performed as follows. Fr-I was incubated at 37°C with 20mg of pronase (Kaken Kagaku Co., Tokyo, Japan) in 10 ml of 2% Triton X-100, 50 mM Tris-HC1 (pH 7.2). After 24 hr, an additional 20 mg of pronase in 1 ml of the buffer were added, and the mixture was incubated for an additional 24hr. The reaction was brought to a stop by the addition of an equal volume of 8 M urea. The radiolabelled materials were then applied onto a DEAE-Sepharose CL-6B (Pharmacia) column (1 x 2 cm) previously equilibrated with 4 M urea, 1% Triton X-100, 25 mM Tris-HCl buffer (pH 7.2). Fractions (0.4 ml) of the material were then collected. After washing the column with 20 ml of the same buffer, stepwise elution with 0.15, 0.3, 0.45, and 2.0 mM NaCI was carried out at a flow rate of 12 ml/hr. The mode of sulfatase preparation was as follows (Tsuji et al., 1980). One gram (wet wt) of corpus or antrum was cut into small pieces, placed in 4 ml of 20 mM Tris-HCl (pH 7.2), and disrupted for 2min in a motor driven

207 CRP

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S. OHARA and K. HOTTA

Potter-Elvehjem Teflon homogenizer. The homogenate was used as the enzyme source. Aliquots of the homogenate were taken for protein measurement. Arylsulfatase activity was assayed with nitrocatechol sulfate as the substrate. The incubation mixture (200#1) contained 40ktmol of sodium acetate acetic acid buffer (pH 5.61, 0 . 6 4 # m o l of substrate, and 81d of enzyme preparation. Following incubation at 37"C, the reaction was brought to a stop by immersing the reaction tube in boiling water for 2 min. The mixture was made clear by centrifugation. An aliquot of the supernatant solution was mixed with 0.1 M N a O H and the a m o u n t of liberated nitrocatecholate ion was estimated spectrophotometrically at 515 nm. The sulfatase activity for 35S-labelled Fr-I was assayed as follows. The incubation mixture (200/d), containing 40 #mol of sodium acetate-acetic acid buffer (pH 5.6), 1000 d p m of 35S-labelled Fr-I, and 8/zl of enzyme preparation, was incubated at 37°C for 40 min and the reaction terminated by immersion in boiling water for 2 min. Ethanol was added to the mixture to a concentration of 75%. The mixture was allowed to stand at 0-'C for 30 min, and centrifuged at 10,000 rpm for 30 rain. The radioactivity of the resulting precipitation and supernatant was measured. Radioactivities were measured in as Mark III scintillation counter (Searle Analytic Co.), with Aquasol-2 (New England Nuclear) as the scintillant. RESULTS The degree of incorporation of [3H]glucosamine a n d [35S]sulfate i n t o t h e g a s t r i c m u c u s g l y c o p r o t e i n is s h o w n in T a b l e s 1 a n d 2. T h e r a d i o a c t i v i t y d i s t r i b u tion o f 3H a n d 3~S i n c o r p o r a t i o n i n t o t h e m u c u s g l y c o p r o t e i n o f t h e tissue a n d m e d i u m r e m a i n e d essentially the same throughout the period of fasting. T w e n t y - o n e - 2 9 % o f t h e 3H r a d i o a c t i v i t y i n c o r p o r a t e d into t h e g l y c o p r o t e i n w a s f o u n d in t h e m e d i u m (after 5 h r i n c u b a t i o n ) . T h e f a s t i n g p e r i o d s p r o d u c e d a slight r e d u c t i o n in t h e d e g r e e o f [ 3 H ] g l u c o s a m i n e incorporation into the corpus and antrum mucus g l y c o p r o t e i n . N o r e g i o n a l d i f f e r e n c e s in i n c o r p o r a t i o n c o u l d be o b s e r v e d . A c o n s i d e r a b l e d e c r e a s e in

0.15 M NaC [

O )<

Table 1. Effects of fasting on [3H]glucosamine incorporation into gastric mucus glycoprotein Mucus glycoprotein of tissue Corpus

0hr 24 hr 72 hr Antrum 0 hr 24 hr 72 hr Mean -+ SEM. *Values are expressed 3 of each group.

Mucus glycoprotein of medium

30.4 + 0.15* 27.2 ± 0.52 25.0 ± 0.26 32.8 _+0.02 24.6 _+0.18 27.0 + 0.05

11.8 ± 0.07* 11.3 + 0.26 9.8 _+0.12 12.3 ± 0.03 8.4 + 0.06 7.2 + 0.02

as dpm per #g of tissue protein. There were

Table 2. Effects of fasting on [3-SS]sulfateincorporation in gastric mucus glycoprotein Mucus glycoprotein of tissue Corpus

0hr 24 hr 72 hr Antrum 0hr 24 hr 72hr Mean + SEM. *Values are expressed 3 of each group.

Mucus glycoprotein of medium

9.94_+0.78* 7.02 -+ 1.32 1.74 + 0.26 1.18-+0.03 0.90 ± 0.02 1.42_+0.36

4.17±0.41" 1.64 + 0.33 0.73 + 0.14 0.78+(I.05 0.29 + 0.08 0.54-+0.16

as dpm per ,ug of tissue proteins. There were

[35S]sulfate i n c o r p o r a t i o n i n t o t h e g l y c o p r o t e i n w a s n o t e d in t h e c o r p u s m u c o s a o f 72 h r f a s t e d rats. Since 3 5 S - i n c o r p o r a t i o n into a n t r a l m u c u s g l y c o p r o t e i n w a s low in t h e u n f a s t e d a n i m a l s , e s t i m a t i o n o f f a s t i n g effects o n a n t r a l m u c u s g l y c o p r o t e i n w a s difficult. DEAE-Sepharose CL-6B column chromatography was carried out on corpus mucus glycoprotein whose r a d i o a c t i v i t y v a l u e s differed c o n s i d e r a b l y m o r e t h a n those of the antrum glycoprotein. Three fractions, 1 u n a b s o r b e d a n d 2 a b s o r b e d , were s e p a r a t e d f r o m t h e c o l u m n as s h o w n in Fig. 1. A t a n N a C l c o n c e n t r a t i o n e x c e e d i n g 0.45 M , n o r a d i o a c t i v i t y c o u l d be d e t e c t e d . The amount of unabsorbed fraction increased with p r o l o n g e d f a s t i n g ( T a b l e 3).

0,3 M

0.45M

2.0M

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NaCI

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60

80

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Fig. 1. DEAE-Sepharose CL-6B column chromatography of pronase treated corpus mucus glycoprotein from a fed rat. A sample was eluted stepwise with NaC1 as indicated.

Effects of fasting on gastric mucus biosynthesis Table 3. Distribution of pronase treated corpus mucus glycoprotein on DEAE-Sepharose CL6B column chromatography Fasting period 3H-labelled glycoprotein 0 hr 24 hr 72 hr "S-labelled glycoprotein 0 hr 24 hr 72 hr

Unabsorbed fraction

0.15M NaCI fraction

0.3M NaCI fraction

12.5" 12.0 17.4

11.9 12.7 4.8

6.0 2.6 3.0

0.71" 1.11 0.48

4.92 5.11 1.09

4.39 0.79 0.17

*Values are expressed as dpm per #g of tissue protein.

Figure 2 shows the effects of fasting on arylsulfatase activity in the corpus and antrum, expressed in mol catecholate ion per hour per mg protein. In the corpus, this activity gradually increased at 0, 24 and 72 hr to 0.406, 0.419 and 0.648, respectively. In the antrum, the activity also increased at 0, 24 and 72 hr to 0.478, 0.498 and 0.572, respectively. However, no sulfatase activity, measured using sulfated mucus glycoprotein as the substrate, could be detected in either the corpus or antrum. DISCUSSION

The proliferation of gastric cells is known to be inhibited by fasting (Willems et al., 1971a, b). Majumdar reported that fasting decreases DNA synthesis and the activity of thimidine kinase and D N A polymerase in the corpus mucosa (Majumdar, 1983). Our previous observations showed gastric mucus glycoprotein to accumulate in the corpus region as a result of prolonged fasting (Ohara et al., 1984). The above data indicate that fasting greatly influences the biologically activity of oxyntic gland cells, since this activity is determined primarily and directly by external stimuli such as the passage of foodstuffs through the intestinal tract. Homeostasis is maintained; that is, depletion of liver glycogen is stimulated by fasting (Stetten et al., 1956) so that the blood sugar level may be kept constant. In this study, an attempt was made to determine whether the increase in mucus glycoprotein during

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0.25

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Fasting Time (hr) Fig. 2. Effects of fasting on the activity of gastric arylsulfatase in the corpus ( S O) and antrum (O O).

209

fasting resulted from acceleration of mucus biosynthesis or suppression of mucus secretion. According to our observations, the increase in mucus glycoprotein was due to the suppression of mucus secretion rather than stimulation of mucus biosynthesis. A similar observation on gastric glycoprotein biosynthesis in fasting rats was made by Dekanski et al. (1975), who reported a reduction in gastric glycoprotein synthesis as measured by the rate of Nacetyl-[3H]glucosamine incorporation. Our results showed the incorporation of glucosamine into mucus glycoprotein fraction to decrease by about 20~. Dekanski's data indicated about a 50~o decrease in glycoprotein synthesis. This difference may possibly stem from differences in experimental procedures. For example, the labelled precursors and labelling time were not the same. If fasting decreases the transport of sugar into gastric cells, the transport of glucosamine should be influenced much more than that of N-acetylglucosamine. The reason for this is that the transport of glucosamine into cells is carrier mediated and the influx of N-acetylglucosamine is a simple process of diffusion (Tesoriere et al., 1972). Thus, data disagreement would not arise from differences in labelled precursors. As for the labelling time, the incubation time of Dekanski was 1 hr but ours was 5 hr. The effects of fasting on biological functions may diminish after several hours in vitro. But with the incorporation of sulfate into the gastric mucus glycoprotein, such effects continue in vitro for as long as 5 hr. Fasting greatly decreased the sulfation of corpus mucus glycoprotein, as was also noted in our previous research (Ohara et al., 1984). This means that the sulfate content of corpus mucus glycoprotein and the HID-AB positivity of corpus mucosa decreased with fasting. Thus the effects of fasting on biological functions may be considered to continue for several hours in vitro. At present, the reason as to why our data and those of Dekanski differ remains unclear. It is clear, however, that fasting does not increase the biosynthetic activity of gastric mucus glycoproteins in any case. Fasting may in some way be related to HC1 and pepsinogen secretion and consequently a factor responsible lbr the decrease in gastric mucus glycoprotein sulfation. This is supported by the fact that sulfated mucus glycoprotein functions to protect the gastric gland components from peptic digestion (Takagaki and Hotta, 1979). Accordingly, to ensure the secretion of HCI and pepsinogen, sulfated mucus glycoprotein must perform this protective role but sulfated mucus glycoprotein is not necessarily required for the temporary termination of HC1 and pepsinogen secretion. Thus, the secretion of HC1 and pepsinogen may be inhibited as a result of fasting, and the sulfation of gastric mucus glycoprotein is inhibited as a result of temporarily terminating its secretion. Confirmation of this point will require further research. By the mechanism for the decrease in sulfated mucus glycoprotein, we investigated sulfatase activity in gastric tissue. There is no sulfatase for sulfated mucus glycoprotein, and consequently, its decrease cannot be attributed to the degradation of sulfated mucus glycoprotein. But fasting does increase arylsulfatase activity. The significance of this is not clear at present. The transport of sulfate into

S. OHARA and K. HOTTA

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cells is carrier mediated a n d activation of sulfate is necessary for the sulfation of mucus glycoprotein. Arylsulfatase m a y thus participate at various points in the process of sulfation, such as the storage of activated sulfate in the cells. This point should be studied in detail. In closing, o u r d a t a show the increase in mucus glycoprotein during fasting n o t to be caused by acceleration of its biosynthetic activity. Fasting results in decreases in the sulfation of corpus mucus glycoprotein. Acknowledgement--This work was supported in part by grants-in-aid from the Japanese Ministry of Education. REFERENCES

Allen A. (1981) Physiology of the Gastroentestinal Tract, 1st edn, Vol. 1, pp. 617-639. Raven Press, New York. Azuumi Y., Ohara S., Ishihara K., Okabe H. and Hotta K. (1980) Correlation of quantitative changes of gastric mucosal glycoproteins with aspirin-induced gastric damage in rats. Gut 21, 533-536. Dekanski J. B., MacDonald A., Sacra P. and Parke D. V. (1975) Effects of fasting, stress and drugs on gastric glycoprotein synthesis in the rat. Br. J. Pharmac. 55, 387-392. Horowitz M. I. (1977) The Glycoconjugates, 1st edn, Vol. 1, pp. 189-213. Academic Press, New York. Majumdar A. P. N. (1983) Regulation of gastric musosal DNA synthesis during fasting and refeeding in rats. Digestion 27, 36-43. Ohara S., Ishihara K., Kakei M., Azuumi Y. and Hotta K. (1982) Distribution of mucosal macromolecular glycoproteins in rat stomach. Comp. Biochem. Physiol. 72B, 309-311.

Ohara S., Ishihara K., Goso K. and Hotta K. (1983) The site of sulfated glycoprotein biosynthesis in rat gastric mucosa. Comp. Biochem. Physiol. 76B, 5 8. Ohara S., Kakei M., Ishihara K., Katsuyama T. and Hotta K. (1984) Effects of fasting on mucus glycoprotein in rat stomach. Comp. Biochem. Physiol. 79B, 325 329. Parke D. V. and Symons A. M. (1979) The biochemical pharmacology of mucus. Adv. exp. Med. Biol. 89, 423-441. Stetten M. R., Katzen H. M. and Stetten D. Jr. (1956) Inhomogeneity of glycogen as a function of molecular weight. J. biol. Chem. 222, 587 599. Takagaki Y. and Hotta K. (1979) Characterization of peptic inhibitory activity associated with sulfated glycoprotein isolated from gastric mucosa. Biochim. biophys. Acta 584, 288 297. Tesoriere G., Dones F., Magistro D. and Castagnetta L. (1972) Intestinal absorption of glucosamine and Nacetylglucosamine. Experientia 28, 770-771. Tsuji M., Nakanishi Y., Habuchi H., Ishihara K. and Suzuki S. (1980) The common identity of UDP-Nacetylgalactosamine 4-sulfatase nitrochatechol sulfatase (arylsulfatase), and chondroitin 4-sulfatase. Biochim. biophys. Acta 612, 373-383. Wang C. S. and Smith R. L. (1975) Lowry determination of protein in the presence of Triton X-100. AnaO~t. Biochem. 63, 414-417. Willems G., Vansteenkiste Y. and Smets P. (1971a) Effect of food ingestion on the cell proliferation kinetics in the canine fundic mucosa. Gastroenterologia 61, 323 327. Willems G., Vansteenkiste Y. and Verbeustel S. (1971b) Autoradiographic study of cell renewal in fundic mucosa of fasting dogs. Acta Anat. 80, 23-32. Winet H. (1976) Ciliary propulsion of objects in tubes. Wall drag on swimming Tetrahymena (ciliate) in the presence of mucin and other long-chain polymers. J. exp. Biol. 64, 283-302.