INTRODUCTION

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PANCREATIC EXOCRINE secretion is mainly regulated by gastrointestinal hormones and the autonomic nervous system. Some nutrients, such as amino acids, affect pancreatic endocrine and exocrine secretion (12). Short-chain fatty acids, which are major end products of microbial fermentation in the alimental canal of herbivores, stimulate pancreatic exocrine secretion (7, 13), although there are differences among species in the response induced by these short-chain fatty acids (11). In sheep, exogenously added short-chain fatty acids not only increase pancreatic juice flow, but also enzyme secretion (13). This increase in enzyme secretion appears to be due to a direct effect on acinar cells and not due to an indirect effect through vagus stimulation.

Whereas the intracellular signaling system for pancreatic exocrine secretion is mediated by an increase in intracellular Ca ion in response to CCK and ACh (27), the secretory process induced by short-chain fatty acids in the ovine pancreatic acinar cells resembles that of ACh and may be mediated by intracellular Ca ions (17). However, the physiological significance of these compounds as pancreatic digestive enzyme stimulators remains questionable, because the peripheral concentration of short-chain fatty acids is very low (4). Recently Tanaka et al. (28) showed the potentiation of CCK-induced amylase release by vasoactive intestinal polypetide (VIP) in guinea pig. Thus the action of low concentrations of short-chain fatty acids on pancreatic exocrine secretion is particularly intriguing.

In the present study, we investigated the action of one of the major short-chain fatty acids, propionate, on carbachol (CCh)-induced amylase release using isolated pancreatic acini from guinea pigs, voles, and mice. Amylase release induced by CCh was potentiated by propionate at concentrations that alone were unable to induce secretion in guinea pig and vole acini. The mechanism of this potentiation does not appear to involve the mobilization of intracellular Ca ions or cAMP.

	METHODS

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Animals and treatments. The Hartley strain of male guinea pigs (300-400 g) was obtained from Tokyo Animal Center, Tokyo, Japan, and fed on a commercial diet (Clea Japan). The male ICR strain of mice (25-30 g) was fed on a commercial diet (Clea Japan), and Japanese field voles (Microtus montebeli; 30-40 g), fed on hay and a commercial diet for herbivores (Oriental Yeast), were bred under standard conditions. Food was withheld for ~24 h before experimentation. In the morning, each animal was anesthetized with 4% halothane and immediately decapitated. Pancreas were rapidly removed and incubated in ice-cold HEPES-buffered Ringer (HBR) solution. All experiments were conducted in accordance with guidelines on the care of experimental animals as approved by the Physiological Society of Japan.

Materials. Materials were purchased from the following sources: bovine serum albumin (BSA; fraction V) from Sigma Chemical (St. Louis, MO), collagenase Type IV from Worthington Biochemicals (Freehold, NJ), trypsin inhibitor (TI) from Sigma Chemical, carbachol from Sigma Chemical, propionate from Nakarai Tesque (Kyoto, Japan), Cell-Tak from Collaborative Biomedical Products (Bedford, MA), soluble Zulkowsky starch from Merck (Darmstadt, Germany).

Isolation of pancreatic acini. Pancreatic acini were isolated from pancreas according to the collagenase digestion procedure described by Tanaka et al. (28). HBR solution (in mM: 24.5 HEPES, 98 NaCl, 6 KCl, 2.5 NaH₂PO₄, 5 sodium pyruvate, 5 sodium fumarate, 5 sodium glutamate, 11.5 glucose, 2 L-glutamine, 1 CaCl₂, 1 MgCl₂, and 1% vitamin solution, 0.2 % BSA, 0.01% TI, pH 7.4) (5 ml) containing purified collagenase (65-75 U/ml) was injected intramurally into isolated pancreas, and the pancreas was incubated at 37°C for 60 min with continuous shaking (120 cycles/min). After the incubation, the pancreatic tissue was triturated five times with a glass pipette (2-mm tip diameter) and filtered through nylon mesh (300 µm lattice). Isolated acini thus obtained were washed three times with enzyme-free HBR and used for measurements of amylase release or intracellular Ca²⁺ concentration ([Ca²⁺]_i).

Perifusion. Isolated acini were resuspended in perifusion HBR solution, transferred to a perifusion chamber (0.95 ml) consisting of a filter (5 µm lattice; Millipore, Bedford, MA) and a filter holder (Millipore), and perifused at a rate of 1 ml/min with a peristaltic pump. The effluent from the filter holder was collected every 2 min and stored on ice for determination of amylase content. LDH leaked into the perfusate from this preparation was ~0.08% of total amounts of pancreatic acini and did not increase by various stimulations (10 µM CCh, 100 µM propionate, and the combination with both substances). Staurosporine (Sigma Chemical) and Ca ionophore A23187 (Sigma Chemical) were added to the perifusion solution when required. DMSO was used as a solvent for staurosporine and A23187. The concentration of DMSO was 0.2% in the perifusion solution. DMSO alone at this concentration had no effect on amylase secretion.

Measurement of amylase release. Amylase activity was assayed by a modified method of Bernfeld using soluble Zulkowsky starch (5) and was expressed as a percentage of the total amylase activity contained in acini.

Measurement of [Ca²⁺]_i. Isolated acini were loaded with 5 µM fura 2-AM (40 min, 37°C) and transferred to a coverslip dish chamber (LU-CSD, Medical Systems). Quantitative fluorescence imaging was performed. The ratio between fluorescence intensities at 510 nm with excitation at 340 and 380 nm was measured from single cells in pancreatic acini according to method described by Kanno et al. (16). [Ca²⁺]_i was calculated from the ratio according to the equation of Grynkiewicz et al. (8). Pancreatic acini were perifused at the rate of 1.5 ml/min, and drugs were added to the perifusion solution. The chamber fluid level was adjusted to 250 µl by an aspirator (LU-ASP, Medical Systems). The half-time for a complete change of the solution in the chamber was ~10 s.

Measurement of cellular cAMP. Cellular cAMP was determined using an enzyme immunoassay system (RPN225, Amersham, Tokyo, Japan). Five guinea pigs were used for the cAMP measurement. Isolated acini from one guinea pig were split into five chambers and perifused at rate of 1 ml/min. The effluent from the filter holder was collected every 2 min and stored on ice for the determination of amylase content. Propionate (100 and 600 µM), CCh (10 µM), and combinations of CCh (10 µM) and propionate (100 µM) were individually added to the solution. All solutions contained 0.1 mM 3-isobutyl-1-methylxanthine (Sigma Chemical). Stimulations were carried out for 10 min. For each assay, a standard curve was constructed by adding known amounts of cAMP to standard incubation solution. Results for cyclic nucleotide content were expressed as percent of control; the control value was obtained from the perifused acini without secretagogues.

Statistics. All data are expressed as means ± SE of several experiments and were analyzed by Student's t-test or Cochran-Cox test. P values of <0.05 were considered statistically significant.

	RESULTS

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Effect of propionate on amylase release from guinea pig acini. It was previously reported that propionate stimulates pancreatic exocrine secretion in guinea pigs in vivo (10). The secretory response of propionate alone was examined in the isolated, perifused guinea pig pancreatic acini (Fig. 1). Amylase release from this preparation was maintained for 40 min at a constant low level <0.2% of total. Continuous administration of 100 µM propionate failed to significantly increase amylase release (P > 0.05). On the other hand, 300 µM propionate increased amylase secretion after 4 min and at 6 min reached levels that were six times higher than control levels. However, this increase was not maintained, but returned to control level after 12 min. Furthermore, administration of 600 µM propionate caused a rapid increase in amylase release, which, after 4 min, was ninefold higher than control values. This secretory response was also transient and rapidly declined, but after 14 min amylase release gradually increased again and was maintained at a level that was threefold higher than control output.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Time course of amylase release induced by graded concentrations of propionate from perifused guinea pig pancreatic acini. Propionate was administered in doses of 100 (b), 300 (c), and 600 µM (d) or saline (a) was administered. Each value represents means ± SE of 4-6 experiments.

Effect of simultaneous stimulation of guinea pig acini with CCh and propionate. At first, the secretory responses of CCh, a stable analog of ACh, were examined in isolated, perifused pancreatic acini of guinea pigs (Fig. 2). CCh evoked a concentration- and time-dependent increase in amylase secretion. A continuous administration of CCh 3 µM caused an increase in amylase release, which reached a peak level (0.32% of total) after 4 min. The administration of 10 µM CCh produced greater amylase release: a 3.4-fold increase after 6 min compared with the control. After that the output declined slightly, but was maintained at a 2.8-fold higher level than control levels during stimulation. With 100 µM CCh, amylase release increased rapidly, reaching a peak value of fivefold (0.85% total) after 4 min. Subsequently, the effect of 100 µM propionate on 10 µM CCh-induced amylase release was examined in guinea pig pancreatic acini (Fig. 3A).

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Time course of amylase release induced by graded concentrations of carbachol from perifused guinea pig pancreatic acini. Carbachol (CCh) was administered in concentrations of 3 (b), 10 (c), and 100 µM (d) or saline (a) was administered. Each value represents means ± SE of 4-6 experiments.

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Time course of amylase release induced by simultaneous administration of CCh and propionate. Amylase release induced by simultaneous administration of CCh and propionate was investigated in guinea pigs (A), voles (B) and mice (C). Responses to saline (a), propionate (100 µM) alone (b), CCh (10 µM) alone (c), and a combination of CCh (10 µM) and propionate (100 µM) (d) are shown. Each value represents means ± SE of 4-6 experiments. * P < 0.05, ** P < 0.01 compared with CCh alone.

Continuous administration of 100 µM propionate did not cause any secretion (Fig. 3Ab). Continuous administration of 10 µM CCh augmented amylase release after 2 min; peak values were attained after 6 min and were maintained at this level during continuous stimulation (Fig. 3Ac). When both 100 µM propionate and 10 µM CCh were administered simultaneously, amylase release increased more rapidly, reached a peak value (0.9% of total) after 6 min, and thereafter gradually declined (Fig. 3Ad). The total 30-min value for amylase release during simultaneous stimulation with CCh and propionate was significantly greater (2.3-fold) than summated values of amylase release evoked by CCh and propionate alone (P < 0.01; Fig. 4A). Thus propionate potentiated CCh-induced amylase release from guinea pig acini.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Effects of propionate on CCh-induced amylase release in guinea pig (A), vole (B), and mouse (C) acini. Each value represents total amount of amylase release minus the control during a 30-min stimulation in acini derived from guinea pigs, voles, and mice. Response to a combination of CCh (10 µM) and propionate (100 µM) was compared with summated value obtained with CCh (10 µM) and propionate (100 µM) alone. Values are means ± SE of 4-7 experiments. ** P < 0.01 compared with summated values of CCh and propionate alone.

Secretory response of vole pancreatic acini. Pancreatic lobules of voles respond to short-chain fatty acids (11). So we examined whether the potentiating action of propionate on CCh-induced amylase release in vole pancreatic acini was similar to that obtained in guinea pig acini.

Figure 3Bb shows that continuous administration of 100 µM propionate did not cause any change in amylase release. Again, CCh 10 µM increased amylase secretion after 2 min, which reached a peak value (0.5% of total) after 4 min and was maintained this level during stimulation (Fig. 3Bc). The simultaneous administration of 100 µM propionate and 10 µM CCh caused a rapid rise in amylase release that reached a peak value (0.75% of total) and was maintained at a slightly lower level (Fig. 3Bd). The total 30-min value for amylase release during simultaneous stimulation with CCh and propionate was significantly greater than the summated values of amylase release elicited by CCh and propionate alone (P < 0.01; Fig. 4B). Thus propionate also potentiated CCh-induced amylase release from pancreatic acini of voles.

Secretory response of mouse pancreatic acini. The potentiating action of propionate on CCh-induced amylase release observed in the herbivorous guinea pig and vole was also investigated in the omnivorous mouse.

Propionate 100 µM failed to alter amylase release in mouse acini as was observed in guinea pig and vole acini in that release was not significantly enhanced (Fig. 3C, a and b). Administration of 10 µM CCh produced a ninefold increase in amylase release after 6 min (Fig. 3Cc). The response then rapidly declined to a level that was twofold higher than control levels. The simultaneous administration with 100 µM propionate and 10 µM CCh produced a secretory response that was comparable to that obtained with CCh alone (Fig. 3Cd). The total value for amylase secretion during simultaneous stimulation with CCh and propionate for 30 min was not significantly larger than the summated values for amylase release elicited by CCh and propionate alone (P > 0.05; Fig. 4C). Thus the potentiating action of propionate was species dependent.

Effect of staurosporine application on the potentiating action of propionate- and CCh-induced amylase secretion in guinea pig acini. The effect of staurosporine on the potentiating action of propionate on CCh-induced amylase release was examined in guinea pig acini. Amylase release induced by CCh was potentiated by 126% by propionate (100 µM), but this potentiating response was unaffected by staurosporine (1 µM) at a concentration known to inhibit protein kinase C (24).

Effect of propionate and A23187 on amylase release in guinea pig acini. It is known that the Ca ionophore A23187, which transports Ca²⁺ into the organic phase, elicits pancreatic amylase release (26). In the present study, the effect of propionate on amylase release induced by Ca ionophore was investigated in the guinea pig pancreatic acini. As shown in Fig. 5, amylase release from guinea pig pancreatic acini was increased 5% by A23187 (5 µM). The simultaneous addition of propionate (100 µM) and A23187 (5 µM) potentiated amylase release about twofold compared with A23187 alone (P < 0.05).

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Effect of propionate on Ca ionophore-induced amylase release in guinea pig acini. Each value represents total amount of amylase release minus control during a 30-min stimulation in acini derived from guinea pig. Response to a combination of Ca ionophore A23187 (5 µM) and propionate (100 µM) was compared with value obtained with A23187 alone. Values are means ± SE of 5 experiments. * P < 0.05 compared with A23187 alone.

Effect of propionate on basal and CCh-stimulated [Ca²⁺]_i in guinea pig acini. It is generally accepted that amylase release by ACh and CCK is mediated by increases in [Ca²⁺]_i. The question as to whether the mobilization of [Ca²⁺]_i contributes to the potentiating action of propionate on CCh-induced amylase release was examined using isolated acini loaded with fura 2-AM.

Propionate 100 µM, which did not elicit an amylase release, failed to produce a significant rise in [Ca²⁺]_i (Fig. 6A). The administration of 300 or 600 µM propionate, which evoked amylase release, also produced no remarkable effects on [Ca²⁺]_i. The average concentration of [Ca²⁺]_i obtained with each dose of propionate (100 and 600 µM) was comparable to that obtained under basal conditions (P > 0.05) (Fig. 7, B and C).

View larger version (30K): [in this window] [in a new window]   Fig. 6.   Representative traces of intracellular Ca2+ concentration ([Ca2+]i) induced by propionate and CCh in guinea pig pancreatic acini. A: changes in [Ca2+]i induced by propionate (100, 300, and 600 µM). B: Changes in [Ca2+]i induced by CCh (1, 3, 10, and 100 µM). Each set of 5 lines was obtained from 1 cluster of pancreatic acini.

View larger version (19K): [in this window] [in a new window]   Fig. 7.   Changes in [Ca2+]i induced by propionate and CCh in guinea pig acini. A: average [Ca2+]i induced by CCh (1, 3, 10, and 100 µM). B: combination of CCh (1 µM) and propionate (100 µM). C: combination of CCh (10 µM) and propionate (600 µM). Each value represents average [Ca2+]i for a 3-min stimulation. Values are means ± S.E. of 7-10 experiments. * P < 0.05, ** P < 0.01 compared with basal values.

Typical examples of changes in [Ca²⁺]_i during exposure to CCh application are shown in Fig. 6B. With 1 µM CCh, [Ca²⁺]_i increased after 10 s and reached at plateau level (~300 nM) after 30 s and gradually returned to resting levels after the removal of CCh. When the concentration of CCh was increased, [Ca²⁺]_i increased dose dependently. However, there was a decreased tendency for [Ca²⁺]_i to rise during stimulation by 100 µM CCh (Fig. 7A).

Simultaneous exposure to 100 µM propionate and 1 µM CCh produced a change in [Ca²⁺]_i comparable with that obtained by 1 µM CCh alone (P > 0.05; Fig. 7B). Furthermore, the effect of 600 µM propionate and 10 µM CCh was similar to that observed with 10 µM CCh alone (P > 0.05; Fig. 7C). Comparable effects were also observed after a 9-min stimulation (data are not shown). Thus the potentiating action of propionate on CCh-induced amylase release was not dependent on the mobilization of [Ca²⁺]_i.

Effect of propionate and CCh on cAMP content in guinea pig acini. The effect of propionate on the cellular cAMP content elicited by CCh in guinea pig acini was also examined. As shown in Table 1, application of each chemical significantly increased slightly the cellular cAMP in the acini under the perifusion for 10 min (P < 0.05). However, the response by propionate did not alter dose dependently. The combination of CCh (10 µM) and propionate (100 µM) also slightly increased cellular cAMP level, but the level was almost the same as that induced by CCh (10 µM) or propionate (100 µM) alone.

	DISCUSSION

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The present experiments provide evidence that propionate, a short-chain fatty acid, not only directly evokes amylase release, but also potentiates CCh-induced amylase release from pancreatic acini of guinea pigs and voles. This potentiation by propionate is species specific, because it was not observed in the mouse pancreatic acini.

Propionate and butyrate are potent stimulators of insulin and glucagon release (2, 3). Ovine exocrine pancreas responds not only to butyrate and propionate but also to acetate (13). It is known that there are species difference in the secretory response induced by short-chain fatty acids, not only in endocrine pancreas (15) but also in exocrine pancreas (13). The exocrine pancreas of sheep and guinea pig responds to acetate, propionate, and butyrate, but the pancreas of mice, cats, and fowls do not (10). Furthermore, pancreatic lobules derived from mice do not respond to these fatty acids at all, although preparations from voles are able to evoke amylase release (11). In the present experiment using the isolated perifused pancreatic acini, propionate evoked amylase release and potentiated CCh-induced amylase release in guinea pig and vole. However, in mice, propionate alone failed to produce a rise in amylase release and was unable to potentiate CCh-induced amylase release. These findings are compatible with the view that pancreatic enzyme secretion induced by short-chain fatty acids occurs only in those species in which short-chain fatty acids can be produced and used as a major energy source (11).

Despite the fact that the secretion of insulin (3, 18, 19) and amylase (11, 13) can be altered by certain short-chain fatty acids, the physiological significance of this action of short-chain fatty acids remains questionable (1, 2, 4) because of very low concentration of short-chain fatty acids present in the peripheral circulation and the ability of short-chain fatty acids to stimulate secretion only at relatively high concentrations. Short-chain fatty acids are cleared rapidly from portal blood by the liver, and their concentrations in peripheral circulation remain extremely low even in fed sheep (4). Although the physiological range of concentration of short-chain fatty acids in portal or peripheral blood in guinea pig and vole remains to be fully defined, concentrations of acetate, propionate, and butyrate in carotid artery (~3.4 mM, ~140 and 65 µM, respectively) have been reported in lactating ewes (29). In the present study, however, a low dose of propionate, which by itself possessed no effect on amylase release, potentiated CCh-induced amylase release in guinea pigs and voles. Thus the potentiating action by short-chain fatty acids on cholinergic control of pancreatic exocrine secretion may be physiologically relevant and a characteristic phenomenon in some herbivores. Most herbivores eat more or less continuously, and thus bile and pancreatic juice may be expected to enter the duodenum at frequent intervals throughout the day (14). Further experiments are necessary to clarify whether the actions of short-chain fatty acids resemble the responses to gut peptides.

There appear to be two independent signal transduction pathways, both of which lead to the stimulation of pancreatic enzyme secretion. CCK and ACh are reported to increase [Ca²⁺]_i in pancreatic acinar cells (27) without changing cytosolic cAMP concentrations (6). VIP and secretin have been shown to increase the concentration of intracellular cAMP (32) without increasing [Ca²⁺]_i (9, 20). Tanaka et al. (28) examined the mechanism underlying the potentiation of VIP on CCK-induced amylase release from guinea pig pancreatic acini and suggested that VIP facilitates a Ca²⁺-dependent process distal to the increase in [Ca²⁺]_i to potentiate CCK-induced amylase release.

In regard to the action of short-chain fatty acids on pancreatic exocrine secretion, Harada and Kato (13) found, using the isolated ovine pancreatic lobules, that butyrate acts directly on acinar cells. Although the precise cellular mechanism of digestive enzyme secretion in response to short-chain fatty acids remains unknown, Katoh and Tsuda (17) showed that the cellular secretory process activated by short-chain fatty acids is qualitatively similar to that induced by ACh and that Ca²⁺ ions might be important mediators for these secretagogues in ovine pancreatic acinar cells. In the present experiment, the mechanisms of the potentiating action of propionate on CCh- and A23187-stimulated secretion and the secretory response to propionate alone were examined using isolated guinea pig pancreatic acini loaded with fura 2-AM. CCh elevated [Ca²⁺]_i in acinar cells, but propionate alone did not cause an increase in [Ca²⁺]_i, despite causing amylase secretion by high dose. Furthermore, propionate did not modify the mobilization of [Ca²⁺]_i elicited by CCh. These results suggest that the secretory response elicited by propionate occurs via a pathway that is distinct from that elicited by CCh and not accompanied by an increase in [Ca²⁺]_i. In addition, A23187-induced amylase release was also potentiated by propionate, suggesting that propionate does not act at the level of the cell membrane.

Changes in ionic calcium and cAMP have been linked as coupling factors between cell excitation and hormonal release (23). Butyrate has been shown to increase cAMP levels in several different cell culture experiments. The action appears to be mediated by the stimulation of adenylate cyclase rather than by an inhibition of phosphodiesterase (22, 25). Recently, Nutting et al. (21) demonstrated that the vasorelaxant actions of short-chain fatty acids might be related to increases in tissue cAMP levels. In this experiment, cAMP content in pancreatic acinar cells increased slightly by propionate application alone, but the level did not alter dose dependently. Although the cAMP content also increased slightly by CCh application, the level did not alter by combination with propionate. Furthermore, it is confirmed that the cAMP content increased by VIP was not changed by combination with propionate (unpublished data). Thus the response of the exocrine pancreas to short-chain fatty acids is different from that of VIP and secretin and is not mediated directly by changes in cyclic nucleotide levels.

It is believed that most of the actions of intracellular messengers are mediated by protein kinases and phosphatases (30). Ionic calcium activates a number of distinct kinases and phosphatases (31). Most of these enzymes involve calmodulin as a Ca²⁺ receptor and a catalytic kinase or phosphatase domain. Staurosporine, which affects protein kinase C and tyrosine kinase activities at the concentration used in this experiment, did not alter the potentiating effect of propionate on CCh-induced amylase secretion. This result suggests that the potentiation of CCh-induced amylase release by propionate is not caused by the staurosporine sensitive protein kinase C activation. Propionate has a potentiating effect on the amylase release not only induced by the CCh, but also by the Ca ionophore A23187. In any event, it is thought that propionate facilitates a Ca²⁺-dependent process distal to the increase in [Ca²⁺]_i to potentiate CCh-induced amylase release in some herbivores.

In conclusion, propionate potentiates CCh-induced amylase release from the isolated guinea pig and vole perifused pancreas, in addition to exerting a direct action on amylase release. Species difference existed in these secretory responses to propionate. The potentiating action of propionate occurs via a secretory pathway that is not associated with the mobilization of intracellular calcium ions or a rising cAMP.

Perspectives