Effects on Digestive Secretions

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Diabetes Causes and Possible Treatments

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I. Summary_

Rat amylin subcutaneously injected into rats dose-dependently inhibits pentagastrin-stimulated gastric acid secretion and protects the stomach from ethanol-induced gastritis. The ED50s for these actions (0.050 and 0.036 mg, respectively) are the lowest for any dose-dependent effect of amylin thus far described, and their similar potencies are consistent with a mechanistic (causal) association. At higher amylin doses, inhibition of gastric acid secretion was almost complete (93.4%). Gastric injury (measured by a subjective analog scale) was inhibited by up to 67%. The observation that effective doses of amylin result in plasma concentrations of 7-10 pM (i.e., within the reported range; Pieber et al., 1994) supports the interpretation that inhibition of gastric acid secretion and maintenance of gastric mucosal integrity are physiological actions of endogenous amylin. The pharmacology of these responses fits with one mediated via amylin-like receptors.

Advances in Pharmacology, Volume 52 Copyright 2005, Elsevier Inc. All rights reserved.

1054-3589/05 $35.00 DOI: 10.1016/S1054-3589(05)52007-6

Rat amylin inhibited CCK-stimulated secretion of pancreatic enzymes, amylase, and lipase by up to ^60% without having significant effect in the absence of CCK. ED50s for the effect were in the 0.1-0.2 mg range, calculated to produce plasma amylin excursions within the physiological range. Effects of informative ligands are consistent with the concept of amylin receptor mediation. Amylin was effective in ameliorating the severity of pancreatitis in a rodent model.

The amylin analog pramlintide inhibited gallbladder emptying in mice as measured by total weight of acutely excised gallbladders.

Amylin inhibition of gastric acid secretion, pancreatic enzyme secretion, and bile secretion likely represents part of an orchestrated control of nutrient appearance. Modulation of digestive function fits with a general role of amylin in regulating nutrient uptake. Rate of ingestion, rate of release from the stomach, and rate of digestion of various food groups appear to be under coordinate control.

II. Gastric Acid Secretion_

A. Background

Complex carbohydrates, proteins, and triglycerides, comprising the three major food groups, are each formed in condensation (water-forming) reactions. Digestion of these foods into absorbable moieties (e.g., monosac-carides, amino acids, and fatty acids) essentially involves the reversal of this process, hydrolysis (Guyton and Hall, 1996b). Gastric acid participates in this action, especially with respect to protein and triglyceride digestion, and may therefore be regarded as a contributor to the aggregate rate of nutrient uptake (Alpers, 1994).

Amylin is the most potent endogenous inhibitor of gastric emptying so far identified in mammals (Gedulin et al., 1996; Young et al., 1996a), being more potent, per molar subcutaneous dose, than other physiological inhibitors of gastric emptying, secretin, cholecystokinin (CCK), glucagon-like peptide-1 (GLP-1), and peptide YY (PYY) (Young et al., 2002). Control of gastric emptying appears to be a physiological function of amylin, occurring at normal plasma concentrations (Pieber et al., 1994; Young et al., 1995). Gut peptides that slow gastric emptying at physiological concentrations also typically inhibit gastric acid secretion (e.g., secretin, MacLellan et al., 1988; Rhee et al., 1991; CCK, Burckhardt et al., 1994; Konturek et al., 1992; GLP-1, Schjoldager et al., 1989; PYY, Adrian et al., 1985; Guo et al., 1987; Pappas et al., 1985, 1986;). In view of this association, and because of the potential role of acid secretion in control of nutrient availability, the effect of amylin on gastric acid secretion is of interest.

B. Effect of Amylin on Gastric Acid Secretion in Rats

The effects of amylin or pramlintide on acid secretion have not been studied in humans. Several methods have been used to study effects of amylin gastric acid secretion in rats. In a modification of the Shay test in rats (Shay et al., 1945), gastric contents were measured 3 hr after pyloric ligation. Amylin injected peripherally in high doses (up to 100 mg/kg) inhibited gastric acid secretion (Guidobono et al., 1994). Such pharmacological doses did not, however, identify this action as physiological. Intracerebro-ventricular administration of amylin was orders of magnitude more potent than intravenous administration in inhibiting gastric acid secretion in a similar preparation (Guidobono et al., 1994).

Studies aimed at determining the physiological relevance of amylin inhibition of acid secretion were performed in rats chronically fitted with double gastric fistulae (Zivic Miller). A grommet-shaped double lumen plug was sutured into the stomach wall, and separate entry and exit cannulae communicating with the gastric lumen were exteriorized at the interscapular region, allowing frequent flushing and assessment of gastric acid secretion by titration of the gastric aspirate. Gastric acid secretion was stimulated with pentagstrin (125 mg/kg s.c.) and followed 40 min later with a range of amylin (or pramlintide doses). Pentagastrin stimulated gastric acid secretion 4.6-fold. Amylin injected 40 min later dose-dependently inhibited gastric acid production by up to 94% with a ty2 of min and an ED50 of 0.05 mg/ rat. In fact, the inhibitory effect was sufficiently profound to reduce acid secretion to approximately one-third of the basal (unstimulated) rate. The ED50 dose was estimated from previously determined pharmacokinetic analyses for this animal model (Young et al., 1996b) to produce a peak plasma amylin concentration of 10 pM (using the relation concentration [pM] = 10a86 log dose in mg + 2.13) and a concentration 60 min after injection of 7 pM, well within the endogenous circulating range. That is, the in vivo dose response for inhibition of gastric acid secretion indicated this was a physiological action of amylin. The in vivo potency of amylin's effect of inhibiting pentagastrin-stimulated gastric acid secretion in the presently described rat model was compared with that of GLP-1 in the same model, and it was 180-fold greater (Gedulin et al., 1997b) (Fig. 1).

C. Localization of Amylinergic Inhibition of Gastric Acid Secretion

Central control of gastric acid secretion involves a cholinergic pathway involving the nucleus tractus solitarius, area postrema, and dorsal motor nucleus of the vagus (Okuma and Osumi, 1986a,b) as well as capsaicin-sensitive vagal afferents (Sakaguchi and Sato, 1987). The area postrema can respond to locally applied agents with changes in gastric acid secretion

Area Postrema Rat
FIGURE 1 Dose response for amylin inhibition of pentagstrin-stimulated secretion of acid from fistulated stomachs of conscious rats. Data from Gedulin et al. (2005).

(Okuma and Osumi, 1986; Okuma et al., 1987; Tache et al., 1989; Zhang and Huang, 1993).

Several authors have concluded that the dominant acid-inhibitory actions of calcitonin gene-related peptide (CGRP) are specific, are central, and are not explained by direct parietal cell effects (Helton et al., 1989; Tache, 1992). As with the effects of infused cholecystokinin and peptide YY to inhibit gastric acid secretion (Lloyd et al., 1997), the effect of CGRP required an intact vagus nerve (Lenz et al., 1985; Tache et al., 1984). Acid-inhibitory effects of centrally administered CGRP were unaffected by systemic antibody that neutralized its peripheral effects (Lenz et al., 1984). These and other studies (Lenz and Brown, 1987; Lenz et al., 1984; Morley et al., 1981; Okimura et al., 1986; Tache et al., 1984, 1991 ) led many to infer a central site of CGRPergic inhibition of gastric acid secretion. As discussed later, central acid inhibitory effects previously ascribed to CGRP are likely to be mediated via an amylin-like pharmacology.

The involvement of central amylin receptors in the control of gastric acid secretion is supported by the dense localization of such receptors in the area postrema/nucleus tractus solitarius (Sexton et al., 1994). This circum-ventricular brain stem structure, which modulates other gastric actions of amylin (Edwards et al., 1998) (described elsewhere), is sensitive to circulating peptides, receives much gastric vagal input (Ewart et al., 1988; Yuan and Barber, 1993), and communicates directly with the dorsal motor nucleus of the vagus (Rogers et al., 1996), whence central acid secretory drive emanates (Guyton and Hall, 1996a). Clementi et al., proposed that the gastroprotec-tive effect of amylin was central and that pathways involved dopamine-2 receptors (Clementi et al., 1996); this proposal was supported by reports that amylin's effect could be blocked with domperidone, a DA2 receptor antagonist (Clementi et al., 1997).

The central effects of amylin in inhibition of gastric acid secretion appeared not to depend upon a somatostatinergic mechanism in the stomach. Pretreatment with cysteamine, which depletes somatostatin in the stomach, did not prevent centrally administered amylin from inhibiting gastric acid secretion in pylorus-ligated rats (Guidobono et al., 1994).

D. Peripheral (Local) Gastric Acid Inhibitory Effect of Amylin

The identification of central mechanisms mediating amylin-inhibition of gastric acid secretion do not preclude the existence of direct preipheral effects. For example, in addition to central mechanisms, secretin inhibits acid secretion through a local effect independently of central connections (Lloyd et al., 1997).

A local somatostatin-dependent action of amylin in inhibiting acid secretion from mouse stomach in vitro was reported (Zaki et al., 1996)

and cannot be excluded as a contributory mechanism. Moreover, this local effect was blocked with AC187, but not CGRP[8-37], indicating it was likely to be specifically amylinergic (Makhlouf et al., 1996; Zaki et al., 1996). In the same preparation, amylin inhibited gastrin secretion as well as somatostatin secretion (Makhlouf et al., 1996).

Past descriptions of possible sites of action of CGRP's inhibition of gastric acid secretion provide clues as to how amylin may operate. Mechanisms have included direct peripheral effects (Holzer et al., 1991; Tache et al., 1991). At the stomach, CGRP is reported to modulate the antral mucosal response to acid (Manela et al., 1995), to locally stimulate somatostatin secretion (Zdon et al., 1988), and to directly stimulate parietal cells (Umeda and Okada, 1987). In regard to a local acid inhibitory effect, it may be significant that amylin-like immunoreactivity in the gut of rats and humans is predominantly in the pyloric antrum (Asai et al., 1990; Miyazato et al., 1991; Mulder et al., 1994; Nicholl et al., 1992; Ohtsuka et al., 1993; Toshimori et al., 1990), where it is localized with gastrin in G-cells (Mulder et al., 1994, 1997; Ohtsuka et al., 1993).

E. Amylin Inhibition of Gastric Acid Secretion During Hypoglycemia

Insulin stimulation of gastric acid secretion (Isenberg et al., 1969) appears to be secondary to its hypoglycemic effect. For example, increases in plasma glucose concentration inhibit gastric acid secretion (Lam et al., 1993; Moore, 1980), including that stimulated by insulin (Stacher et al., 1976). Increases in glucose also inhibit amino acid-stimulated acid secretion (Lam et al., 1995). Studies using microinjection of D-glucose into different brain regions indicate that glucose-induced inhibition of gastric acid secretion appears to be localized to structures around the nucleus tractus solitarius (Sakaguchi and Sato, 1987). Amylin inhibition of gastric acid secretion was not associated with (explained by) changes in plasma glucose concentration (Gedulin et al., 1997b).

Several amylinergic effects, for example, inhibition of gastric emptying (Gedulin and Young, 1998; Gedulin et al., 1997c) and inhibition of glucagon secretion (Parkes et al., 1999), are overridden by hypoglycemia. These patterns suggests "fail-safe" glucose counterregulatory reflexes in which the restraint that amylin exerts on nutrient availability is lifted during hypoglycemia. Whether hypoglycemia overrides amylinergic inhibition of gastric acid secretion has not been directly addressed, although there are clues from the literature that such a mechanism indeed exists.

In a Shay test of gastric acid secretion in pylorus-ligated rats, Guidobono et al. (Guidobono et al., 1994) compared the acid inhibitory effect of intra-cerebroventricular amylin in saline-treated rats with that in rats administered 1U of insulin intravenously. Whereas amylin inhibited acid secretion by 87%

in saline-treated rats, its inhibition of insulin-stimulated acid secretion was much attenuated (27% inhibition). Indeed, acid secretion in the presence of both insulin and amylin was 2.2-fold greater than basal. Although plasma glucose was not reported, the 1 U intravenous dose is likely, from historical responses, to have produced hypoglycemia. That is, as with other central amylinergic responses, amylin inhibition of gastic acid secretion may also be overridden by hypoglycemia.

F. Gastroprotective Effect of Amylin

Amylin at elevated doses was reported to protect against erosions and mucosal damage in rats administered ethanol, indomethacin (Guidobono et al., 1997), reserpine, and serotonin (Clementi et al., 1997). Its gastroprotective effect in those studies was not explored at doses that would mimic physiological fluctuations in plasma concentration. One study reported a gastroprotective effect only when amylin was given centrally, and not when given subcutaneously at doses of 10 and 40 mg/kg (Guidobono et al., 1997). The authors discounted a mechanistic link between acid-inhibitory and gastroprotective effects. Other work reported here found that the dose responses for these two actions were indistinguishable, and thus could well support a causal association.

One study (Clementi et al., 1997) reported gastroprotective effects of amylin with doses likely to result in plasma amylin concentrations of nM, around 100-fold higher than concentrations of endogenous amylin in rats (Pieber et al., 1994; Vine et al., 1998).

G. Physiological Relevance of Amylin Gastroprotection

In a study designed to probe the physiological relevance of amylin in maintenance of the gastric mucosa, fasted male rats were administered various s.c. doses of amylin 20 min before gavage with 1 ml absolute ethanol (Gedulin et al., 1997a). Thirty minutes later, their stomachs were excised and the everted mucosae were immediately graded for severity of mucosal damage by observers blinded to the experimental treatment. They used scores of 0 (no observable damage) to 5 (100% of mucosal surface covered by hyperemia, ulceration, or sloughing), comparable to those developed by Guidobono and others (Guidobono et al., 1997). Amylin given 5 min before the ethanol gavage profoundly and potently protected the stomach from mucosal injury. The injury score was reduced 67%, and the ED50 (0.036 mg/rat) was statistically indistinguishable from that obtained for inhibition of gastric acid secretion. The ED50 dose was estimated to have resulted in a peak plasma amylin concentration of 8 pM (Gedulin et al., 1997a) (that is, within the physiological range). It is therefore possible that endogenous amylin may play a role in the maintenance of gastric mucosal integrity (Fig. 2).

Amylin Secrtion

FIGURE 2 Dose response for gastroprotective effect of amylin in rats gavaged with ethanol. Data from Gedulin et al. (1997a,b).

FIGURE 2 Dose response for gastroprotective effect of amylin in rats gavaged with ethanol. Data from Gedulin et al. (1997a,b).

A physiological role of endogenous hormones has often been inferred from events following their negation. Treatment of rats with the b-cell toxin streptozotocin results in amylin deficiency. Streptozotocin is reported to induce gastric mucosal lesions in rodents (Goldin et al., 1997; Hung and Huang, 1995; Piyachaturawat et al., 1991; Takeuchi et al., 1997). This condition is not reversed by insulin replacement (Piyachaturawat et al., 1991) and therefore appears unlikely to be due to an absence of insulin. In explaining these findings, some have proposed that streptozotocin may be directly toxic at the gastric mucosa. However, such a mechanism does not explain why NOD (non-obese type 1 diabetic) mice with autoimmune b-cell destruction also exhibit gastric erosions (Nishimura et al., 1983). The absence of a factor from the b-cell, such as amylin, could underlie the propensity of both of these models to gastric erosion. The susceptibility of amylin-deficient adult humans to gastric injury is unclear. Type 1 diabetic children, however, have a 3- to 4-fold elevation in rate of peptic disease (Burghen et al., 1992).

An indirect probe of whether endogenous amylin exerted a gastropro-tective effect would be to examine the effects of amylin secretagogues, such as glucose. Prior administration of 0.25 g D-glucose, shown to increase endogenous plasma amylin concentrations in fasted Sprague Dawley rats to 5 pM (Vine et al., 1998), significantly decreased blinded gastric injury score (by 19 ± 5%, P < 0.0005; Gedulin et al., unpublished). One interpretation of this result is that glucose-stimulated endogenous amylin could be protective.

H. Pharmacology of Acid-Inhibitory and Gastroprotective Effects

The literature on the effects of structurally related peptides assists in the interpretation of the pharmacology of amylin-mediated effects on gastric acid secretion and gastric injury. Calcitonin gene-related peptide (Beglinger et al., 1988; Holzer et al., 1991; Lenz and Brown, 1987; Lenz et al., 1984; Okimura et al., 1986; Tache et al., 1991; Zanelli et al., 1992) and teleost calcitonins (eel and salmon) (Doepfner, 1976; Guidobono et al., 1991; Okimura et al., 1986) are reported to inhibit gastric acid secretion and gastric lesions with high potency. When directly compared, they were found to be generally more potent than mammalian calcitonins (Lenz and Brown, 1987; Okimura et al., 1986).

Amylin administered i.c.v. (Guidobono et al., 1994) was more potent than CGRP in the same model (Hughes et al., 1984). The pharmacology, in which the gastric-inhibitory potency of teleost calcitonins = amylin > CGRP > mammalian calcitonins, fits that described for amylin receptors (Beaumont et al., 1993) and cannot accommodate a purely CGRP-like pharmacology; CGRP receptors are not significantly activated by teleost or mammalian calcitonins (Beaumont et al., 1993). An observation that CGRP [8-37] (a CGRP antagonist; Chiba et al., 1989) reverses inhibition of gastric acid secretion (Clementi et al., 1997), does not identify this as a CGRPergic action; at appropriately high doses, CGRP[8-37] can also block amylinergic responses (Young et al., 1992). Instead, blockade of acid inhibitory and gastroprotective effects with AC187 would indicate that this mechanism was likely to be mediated via amylin- or calcitonin-like receptors, since AC187 is 500-fold more selective for amylin versus CGRP receptors, and 25-fold more selective versus calcitonin receptors (Beaumont et al., 1995). Pre-administration of AC187 (3 mg i.v.) negated the gastroprotective effect of rat amylin (0.3 mg s.c.) in ethanol-gavaged rats (87% of control injury score versus 34% in amylin-treated rats). In separate experiments, pre-administration of AC187 negated amylin inhibition of pentagastrin-stimulated acid secretion (Gedulin et al., 2005) (Fig. 3).

III. Pancreatic Enzyme Secretion_

Exocrine secretion of digestive enzymes from the pancreas could be a further determinant of rate of nutrient uptake from meals and was therefore examined as a potential control point in amylinergic influence on nutrient assimilation. Effects of amylin on exocrine secretion of pancreatic enzymes

Amylin Secrtion

Saline Amylin 0.3 /yg AC187+ AC187 Amylin 0.3 jjg alone

FIGURE 3 Reversal of gastroprotective effect of amylin in ethanol-gavaged rats with the selective amylin antagonist AC187. Data from Gedulin et al. (2005).

Saline Amylin 0.3 /yg AC187+ AC187 Amylin 0.3 jjg alone

FIGURE 3 Reversal of gastroprotective effect of amylin in ethanol-gavaged rats with the selective amylin antagonist AC187. Data from Gedulin et al. (2005).

were examined in vivo in rats and in vitro in isolated pancreatic acini and the pancreatic acinar cell line Ar42j. To date, no in vivo studies of amylin actions in this system have been conducted in humans.

A. Effects of Amylin on Pancreatic Exocrine Secretion In Vivo

One study has investigated effects of amylin on pancreatic enzyme secretion in intact rats (Gedulin et al., 1998). The pancreatic duct was cannulated under anesthesia, and secretions were collected every 15 min for assay of amylase and lipase activity, as well as for measurement of secreted volume. Effects of cholecystokinin octapeptide (CCK-8; 1 mg s.c.) or rat amylin alone (0.1-1 mg s.c.) were assessed. CCK-8 increased 60-min secretion of amylase and lipase activity 7.7- and 6.4-fold over basal, respectively. Two-thirds of this increase was attributable to an increased flow, and one-third to an increased enzyme concentration in the secretion. In contrast, amylin had no significant effect on unstimulated enzyme secretion.

When amylin was administered in association with CCK-8, secretion of amylase was suppressed by up to 58%, two-thirds of which was attributable to a reduction in secretory flow, and one-third to a reduction in enzyme concentration. A similar decrease was observed in secreted lipase (Fig. 4).

ED50s for the inhibition of CCK-stimulated juice flow, amylase secretion, and lipase secretion were 0.11 mg, 0.21 mg, and 0.11 mg, respectively.

These ED50s were not statistically different from each other and were calculated from separate kinetic studies (Young et al., 1996b) to have resulted in peak plasma concentrations of 15-26 pM, comparable to the 9 pM (Vine et al., 1998) to 15 pM (Pieber et al., 1994) range reported to circulate in fed rats. This potency is consistent with the concept that inhibition of pancreatic enzyme secretion is a physiological effect of amylin.

Pramlintide produced similar effects on CCK-stimulated pancreatic enzyme secretion.

B. Pharmacology of Exocrine Inhibitory Action of Amylin

As with some other amylinergic responses, the literature on the effects of structurally related peptides (CGRP and teleost and mammalian calcitonins) can assist in the interpretation of the pharmacology of amylin-mediated effects on exocrine pancreatic secretion. The inhibition by amylin of stimulated secretion of pancreatic enzymes is similar to patterns reported for both CGRP (Bunnett et al., 1991) and calcitonins (Funovics et al., 1981; Hotz et al., 1977; Nakashima et al., 1977; Nealon et al., 1990), including salmon calcitonin (Paul, 1975). Stimulated (Mulholland et al., 1989; Nakashima et al., 1977) but not basal (Funovics et al., 1981) secretion was inhibited with calcitonin or CGRP. The observation that effects of CGRP and calcito-nin are additive (Nealon et al., 1990) could be consistent with their acting via a common receptor. If so, this could not be at CGRP receptors, since calcitonins do not significantly interact with them. But CGRP and, especially, salmon calcitonin interact with amylin receptors (Beaumont et al., 1993). These previously reported effects of CGRP and calcitonins to inhibit stimulated pancreatic exocrine secretion in vivo would instead support an effect mediated via an amylinergic pathway.

C. Effects of Amylin in Ar42j Cells

Ar42j cells, a model of pancreatic acinar cells derived from a pancreatic carcinoma line, exhibit many aspects of pancreatic acinar behavior, including secretion of amylase in response to stimulation with pituitary adenylate cyclase activating peptide (PACAP38) (Kashimura et al., 1993; Raufman et al., 1991; Schmidt et al., 1993). PACAP38-mediated signaling in acinar and Ar42j cells appeared to occur via other than cAMP (Kashimura et al., 1993), and is now recognized as occurring via second messengers activation of phospholipase C and mobilization of intracellular calcium (Barnhart et al., 1997). Using the response to PACAP27 and PACAP38 (125 nM) as positive controls (to indicate that cells and signaling pathways were intact), effects of amylin on phospholipase C activation were tested in Ar42j cells. Ar42j cells grown to confluence were incubated overnight with [2-3H]-myo-inositol and

Amylin Secrtion

then for an initial 20 min before exposure to PACAP27, PACAP38, and rat amylin. Inositol phosphates were extracted (Pittner and Fain, 1989), and rates of inositol monophosphate production were assessed as a measure of phospholipase C activation (Young et al., 2005). Although Ar42j cells responded to PACAP27 or PACAP38 with ^4-fold increases in phospholi-pase C activity, there was no significant change in activity following application of rat amylin (1 mM) (Young et al., 2005). These results indicated that effects on exocrine secretion were indirect (e.g., centrally mediated). Although Huang et al., (Huang et al., 1996) reported effects of amylin in Ar42j cells, they saw no effects with CGRP or salmon calcitonin, which would tend to support the absence of an amylinergic effect (Fig. 5).

To probe whether amylin had an effect on signaling pathways other than phospholipase C, responses were assessed in a microphysiometer. Rates of acid production, as measured with a cytosensor microphysiometer (Molecular Devices, Menlo Park, CA) can be used as an indicator of general cellular response, independent of knowledge of a second messenger system (Owicki et al., 1990; Parce et al., 1990; Pitchford et al., 1995). Using the acidification rate response to PACAP38 (125 nM) to verify that cell signaling was intact, the effect of amylin (1 mM) was tested in Ar42j cells. While exposure to PACAP38 for 6 min evoked a characteristically prolonged activation of Ar42j cells, with activity increasing to 215% of basal, the activity following application of rat amylin over the same period was 89% of basal. That is, there was no direct cellular activation by amylin in this cell line.

D. Effects of Amylin in Isolated Acinar Cells

Effects of signaling molecules in derived cell lines can be misleading if such cell lines do not contain the full complement of biologies as the tissues they are purported to imitate. Effects of amylin were tested in primary pancreatic acini isolated by collagenase digestion methods (Amsterdam and Jamieson, 1974; Gardner and Jackson, 1977). Resulting dispersed acini were suspended in agarose and entrapped onto a microphysiometer capsule (Molecular Devices). Isolated acini exposed to PACAP38 (100 nM) for 10 min, used as a positive control, increased their activity to 163% of basal. Exposure to the same concentration of rat amylin for the same period of time had no significant effect (88% of basal activity) (Young et al., 2005).

Fehmann et al., (Fehmann et al., 1990) and Kikuchi et al., (Kikuchi et al., 1991) affirmed that amylin had no direct effect on isolated pancreatic acini, as assessed by release of amylase in vitro. To the extent that Ar42j cells mimic pancreatic acinar cells, there are four independent findings that support the conclusion that pancreatic acinar cells are not amylin responsive.

FIGURE 4 Dose response for amylin inhibition of CCK-stimulated secretion of amylase and lipase from catheterized pancreas in anesthetized rats. Data from Young et al. (2005).

Amylin Secrtion
-A-1 /iM rAmylin
Amylin Secrtion
FIGURE 5 Absence of effect of amylin Ar42j cells on acidification rate, a generalized response (upper panel), or on phosphoinositol turnover (lower panel). In each case, the positive control response to PACAP was present. Data from Young et al. (2005).

In one study, when both were measured, CGRP inhibited CCK-stimu-lated pancreatic enzyme secretion in vivo but not in isolated perfused pancreas or dispersed acini (Mulholland et al., 1989). Those findings pointed to an extrapancreatic (central) control that has been interpreted as vagal cho-linergic (Owyang, 1994).

The report that amylin inhibits enzyme secretion in vivo concurs with the literature for calcitonins and CGRP, and in the simplest interpretation, points to an amylin-like pharmacology. The report that amylin inhibits stimulated pancreatic exocrine secretion in vivo but has no detectable effect in vitro in pancreatic acini or a derivative cell line is consistent with the parallel literature for CGRP. Both literatures are consistent with a central (indirect) amylinergic control of pancreatic enzyme secretion.

E. Physiological Implications of Modulating Enzyme Secretion

Amylin modulation of digestive enzyme secretion aligns with a general effect of regulating digestive function (as exemplified by influence on gastric acid secretion). This general effect further fits with an overall physiological role to regulate nutrient assimilation and rate of glucose appearance. Because surgical patients tolerate excision of large fractions of absorptive gut remarkably well, without obvious malabsorption, many have presumed that digestive and absorptive capacity is present in abundant excess. It may therefore be questioned whether the 24-67% suppression of pancreatic enzyme secretion obtainable with amylin agonists appreciably contributes to control of nutrient assimilation. An attempt to quantify limiting fluxes in absorption (Weber and Ehrlein, 1998) examined the kinetics of absorptive capacity of hydrolysates of each of the major food groups and enabled calculation of the fraction of available gut length required for complete absorption of nutrient entering at the prevailing rate of gastric emptying. Even when prior hydrolysis eliminated intraluminal digestion as a rate-limiting step, at least 55% of gut length was required (indicating only an 80% reserve), dispelling the notion that absorptive capacity is present in great excess. Decreases in rate of intraluminal digestion (by decreasing enzyme activity in the lumen, for example) can only decrease this reserve. A separate report showed that decreases of exocrine secretory capacity to one-third of normal were sufficient to cause steatorrhea (Cole et al., 1987) and affirmed that digestive and absorptive capacity is not in great excess. Indeed, although safety factors (the relation between digestive/aborptive capacity and load) are initially high in suckling rat pups, they approach 1.0 as individuals enter adulthood (O'Connor and Diamond, 1999). These studies (Cole et al., 1987; O'Connor and Diamond, 1999; Weber and Ehrlein, 1998) support the notion that digestive enzymes are secreted parsimoniously, in amounts titrated to be just sufficient for complete digestion and absorption.

Not only does digestive capacity seem parsimoniously distributed, pro-teolytic activity secreted from the pancreas is subject to feedback control, and by an interesting mechanism. Luminal CCK-releasing factor (LCRF), a peptide secreted from proximal gut, presumptively acts at (as-yet-unidentified) intraluminal receptors to amplify the release of CCK from I-cells at the duodenal and jejunal mucosa. Increased CCK secretion thence stimulates pancreatic enzyme secretion, which then digests LCRF and decreases its signal. If there is insufficient protease to digest and neutralize LCRF, more CCK (and protease) is secreted (Spannagel et al., 1996).

These examples illustrate that modulating digestive capacity may have physiological significance as an influx effector in control of fuel balance. A pharmacological example is provided by the clinical experience with enzyme inhibitors. Slowing digestion and absorption of complex carbohydrates by blocking a-glucosidase lowers glycemic indices (Balfour and McTavish, 1993; Coniff et al., 1995), and inhibiting pancreatic lipase results in weight loss (James et al., 1997). Pramlintide is reported to lower plasma glucose (Ratner et al., 1998; Rosenstock et al., 1998) and body weight (Whitehouse et al., 1998) in type 1 and type 2 diabetic patients. The extent to which effects on gut amylase and lipase activity contribute to these effects is not presently established. Such effects, if present in humans, differ from those of irreversible digestive enzyme inhibitors in that they are not associated with an increased incidence of side effects such as flatulence and steatorrhoea.

F. Effects of Amylin on Experimental Pancreatitis in Mice

Agents that inhibit pancreatic enzyme secretion, for example, somato-statin, have the potential to limit severity of disease in acute pancreatitis, a severe condition that in the United States has a prevalence of ^0.5% and claims ^4000 lives annually (Greenberger et al., 1991). In a mouse model of pancreatitis, a frog skin CCK agonist, caerulein, was injected (0.01 mg i.p.) on three occasions, 2 hr apart, and blood was taken 5 hr later for measurement of amylase as an assessment of pancreatic damage (Warzecha et al., 1997). The 2.6-fold elevation in amylase in saline-treated control mice was dose-dependently ameliorated with amylin (0.1 mg doses and above) injected 5 min before the caerulein (Fig. 6).

CGRP was also effective in a caerulein-induced model of pancreatitis (Warzecha et al., 1997). In a study of 94 patients with pancreatitis, salmon calcitonin significantly improved pain and normalization of serum amylase (Goebell et al., 1979). The concordance of effects of amylin, salmon calcito-nin, and CGRP is consistent with the involvement of an amylin-like pharmacology in the amelioration of pancreatitis.

IV. Effects of Amylin on Gallbladder Contraction_

Amylin control of nutrient appearance includes regulation of several digestive functions, including some (acid and lipase secretion) that affect digestion and absorption of fats. In addition to these latter secretions, digestion and absorption of dietary fats are also influenced by secretion of bile into the intestinal lumen following contraction of the gallbladder. Rats do not possess a gallbladder and are thus unsuited for studies of this mechanism. However, mice have a gallbladder, and control of emptying can be studied by comparing weights of acutely excised gallbladders, bile included (Bignon et al., 1999).

Amylin Secrtion

Amylin dose (¿/g) given 5min before 0.01 //g/kg of caerulein

FIGURE 6 Amylin inhibition of caerulein-induced pancreatitis (assessed by plasma amylase concentration) in mice. Data from Young et al. (2005).

Amylin dose (¿/g) given 5min before 0.01 //g/kg of caerulein

FIGURE 6 Amylin inhibition of caerulein-induced pancreatitis (assessed by plasma amylase concentration) in mice. Data from Young et al. (2005).

Following food deprivation for 3 hr, mice were injected s.c. with saline or CCK-8, with or without various s.c. doses of pramlintide. Thirty minutes later, mice were euthanized by cervical dislocation and the gallbladder was excised and weighed. CCK-8 itself evoked gallbladder contraction, as inferred by a 77% decrease in gallbladder weight. Pramlintide alone dose-dependently inhibited gallbladder emptying, as inferred by a doubling in weight of the gallbladder plus bile. The effect of pramlintide (10 mg) to inhibit gallbladder emptying was reversed with co-administration of the selective amylin antagonist AC187 (300 mg s.c.), pointing to an amylin-like pharmacology. Pramlintide did not prevent CCK-stimulated emptying of the gallbladder (Gedulin et al., in press) (Fig. 7).

CGRP infusions in guinea pigs are reported to inhibit CCK-induced gallbladder contraction (Hashimoto et al., 1988) and can cause relaxation of gallbladder smooth muscle in vitro (Hashimoto et al., 1988; Kline et al., 1991). CGRP also inhibited CCK-induced and meal-induced gallbladder contraction in conscious beagle dogs (Lenz et al., 1993). CGRP halved bile

Cgrp Muscle

FIGURE 7 Effect of the amylin agonist pramlintide on inhibition of gall bladder emptying (assessed as gall bladder weight) in mice. The effect was blocked with the selective amylin antagonist AC187. Unpublished data from Gedulin et al.

FIGURE 7 Effect of the amylin agonist pramlintide on inhibition of gall bladder emptying (assessed as gall bladder weight) in mice. The effect was blocked with the selective amylin antagonist AC187. Unpublished data from Gedulin et al.

flow into the duodenum in pigs (Rasmussen et al., 1997) via a cholinergic, CCK-independent mechanism.

In human studies, salmon calcitonin potently inhibited meal-induced contraction of the gallbladder (Jonderko et al., 1989b), increasing interdigestive volume (Jonderko et al., 1989a). Salmon calcitonin had no direct effect in vitro on guinea pig gallbladder contraction (Portincasa et al., 1989), consistent with an extrapancreatic autonomic effect. Concordance of the relaxive effects of amylin agonists CGRP and salmon calcitonin and annulment of the effect with the selective amylin antagonist AC187 suggests that these actions are mediated via an amylin-like pharmacology.

Effects of amylin agonists to inhibit bile ejection are similar to those described for PYY (Hoentjen et al., 2001), which is similarly CCK-independent and is proposed to be vagally mediated.

Hyperglycemia causes a reduction in gallbladder contraction in healthy individuals, via a mechanism that is distinct from CCK (De Boer et al., 1993, 1994). Hyperglycemic reduction of gallbladder contraction was absent in subjects with type 1 diabetes (De Boer et al., 1994). The absence of modulation of contraction was associated with a similar absence of modulation of vagal activity (as inferred from pancreatic polypeptide measurements) (De Boer et al., 1994). An amylinergic mechanism, acting via the vagus as it does for other responses, could partly underlie the inhibition by hyperglyce-mia of meal-induced gallbladder contraction. The absence of amylin could similarly account for the absence of hyperglycemic effect on contractility in type 1 diabetic individuals.

Physiologically, control of bile ejection is one of the cascade of controls that moderates nutrient assimilation from the meal. For agents that physiologically restrict nutrient appearance, Ra (e.g., amylin and PYY; Hoentjen et al., 2001), it is fitting that in addition to limiting other digestive secretions, they also limit bile ejection. Conversely, for agents that enhance Ra (e.g., glucagon), it is similarly consistent that they additionally augment gallbladder contraction (Jansson et al., 1978).

V. Effects of Amylin on Intestinal Glucose Transport_

A further mechanism by which nutrient assimilation might be controlled is control of absorption, independent of effects on gut motility or digestion. While nutritional state can affect brush border enzyme and transporter expression, the evidence that this function can be acutely controlled is sparse. There is one report that insulin may affect this process (Argiles et al., 1992). The possibility that amylin might modulate glucose transport from the gut lumen was tested in an in situ gut loop preparation in anesthetized rats in which the vascular supply was intact but a 25 cm section of jejunum was exteriorized to enable perfusion of the lumen. Phloridzin, an

FIGURE 8 Absence of effect of intravenously infused amylin on uptake of labeled glucose from the in situ perfused gut lumen in anesthetized rats. Plasma glucose was clamped during gut perfusion. Inhibition of glucose uptake was inhibited in the presence of phloridzin, a positive control. Data from Young and Gedulin (2000).

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