Apolipoprotein A-IV (apo A-IV), a peptide expressed by enterocytes in the mammalian small intestine and released in response to long-chain triglyceride absorption, may be involved in the regulation of gastric acid secretion and gastric motility. The specific aim of the present study was to determine the pathway involved in mediating inhibition of gastric motility produced by apo A-IV. Gastric motility was measured manometrically in response to injections of either recombinant purified apo A-IV (200 μg) or apo A-I, the structurally similar intestinal apolipoprotein not regulated by triglyceride absorption, close to the upper gastrointestinal tract in urethane-anesthetized rats. Injection of apo A-IV significantly inhibited gastric motility compared with apo A-I or vehicle injections. The response to exogenous apo A-IV injections was significantly reduced by 77 and 55%, respectively, in rats treated with the CCK1 receptor blocker devazepide or after functional vagal deafferentation by perineural capsaicin treatment. In electrophysiological experiments, isolated proximal duodenal vagal afferent fibers were recorded in vitro in response to close-arterial injection of vehicle, apo A-IV (200 μg), or CCK (10 pmol). Apo A-IV stimulated the discharge of duodenal vagal afferent fibers, significantly increasing the discharge in 4/7 CCK-responsive units, and the response was abolished by CCK1 receptor blockade with devazepide. These data suggest that apo A-IV released from the intestinal mucosa during lipid absorption stimulates the release of endogenous CCK that activates CCK1 receptors on vagal afferent nerve terminals initiating feedback inhibition of gastric motility.
- mesenteric lymph
apolipoprotein a-iv (apo A-IV) is a peptide expressed by the mammalian small intestine and is released in response to triglyceride absorption (23). Apo A-IV has a number of putative roles, including an involvement in chylomicron formation, prevention of atherosclerotic lesions, reverse cholesterol transport, and also as an antioxidant (23). In addition, administration of exogenous apo A-IV has been shown to decrease food intake in rodents (2) and to modulate gastrointestinal (GI) function, including inhibition of gastric acid secretion (17) and inhibition of gastric motility (16).
Apo A-IV synthesis in enterocytes is rapidly increased by intestinal lipid absorption where it is incorporated into nascent chylomicrons within the enterocytes and is secreted along with chylomicrons (23). After exocytosis from enterocytes, apo A-IV dissociates from chylomicrons and circulates as free protein in plasma. There is considerable evidence that apo A-IV is actively involved in intestinal lipid absorption; synthesis and secretion of apo A-IV increases after fat ingestion or intestinal perfusion of lipid in rodents (6, 10). Additional evidence has suggested that chylomicron assembly and transport may be the specific signal for apo A-IV production within enterocytes (2, 24).
However, in addition to its effects on lipid absorption and handling, there is evidence that apo A-IV may have a role in the regulation of food intake and postprandial gastrointestinal function. A role for apo A-IV in the regulation of food intake was first suggested by studies showing that intravenous injections of mesenteric lymph, collected from donor rats actively absorbing lipid and thus containing chylomicrons, was able to inhibit food intake in recipient rats (2). This inhibition of food intake was shown to be independent of the lipid content of chylous lymph and was abolished by immunoneutralization of apo A-IV, but not apo A-I, a structurally related apolipoprotein. Administration of apo A-IV at the dose of 200 μg, an amount comparable to that found in 2 ml chylous lymph, also significantly depressed food intake (3). It was concluded that apo A-IV may be acting within the central nervous system to inhibit food intake, since it was considerably more potent after central administration (3). There is evidence that apo A-IV may also be involved in the regulation of gastrointestinal function. Central injection of apo A-IV inhibits gastric acid secretion and decreases gastric ulcer formation (15). Central injection of apo A-IV also inhibits gastric motility (16), but the role of apo A-IV in regulating postprandial gastric secretory and motor function and the pathways by which it mediates its action are unclear.
We have been interested in the mechanism by which long-chain triglycerides in the intestinal lumen initiate feedback inhibition of gastric function. We have previously demonstrated that inhibition of gastric emptying in response to intestinal perfusion of long-chain triglyceride is dependent on chylomicron formation (19). Further evidence for a role of chylomicron formation comes from experiments in which lymph collected from donor rats actively absorbing lipid inhibited gastric motility in recipient rats, an effect that could not be accounted for by the lipid content of the lymph (4). Furthermore, inhibition of chylomicron formation in the donor rats rendered the lymph ineffective to alter gastric motility in recipient rats (4). More recently, we have shown that inhibition of gastric motility in response to chylous lymph is mediated by a CCK1 receptor and vagal afferent pathway (5). Moreover, chylous lymph can increase CCK-responsive vagal duodenal afferent fiber discharge, an effect that is inhibited by CCK1 receptor blockade (5). These results suggest that a component of lymph, possibly apo A-IV, is an important mediator in the sensory transduction pathway by which vagal afferents respond to intestinal lipid and thus inhibit postprandial gastrointestinal function. During active lipid absorption, there is an increase in the amount of apo A-IV in lymph (21).
In the present study, we tested the hypothesis that apo A-IV stimulates vagal afferent terminals within the wall of the duodenum, resulting in the activation of a vagal afferent, CCK1 receptor-dependent reflex that decreases gastric motility. In these experiments, we determined the effect of apo A-IV on gastric motility in anesthetized rats and examined the role of CCK1 receptors and the vagal afferent pathway using devazepide and functional deafferentation with capsaicin, respectively, in mediating the inhibitory effects of apo A-IV on gastric motility. To confirm the direct involvement of vagal afferent fibers, we recorded duodenal vagal afferent fiber discharge in response to CCK and apo A-IV.
Male Sprague Dawley rats (260–280 g, Harlan Industries, San Diego, CA) were maintained on regular laboratory rodent chow and housed under controlled conditions of illumination (12:12-h light-dark cycle starting at 0700), humidity, and temperature (21°C). Rats were fasted overnight but allowed water ad libitum before all surgical and experimental procedures. The institutional guidelines for the care and use of laboratory animals were followed throughout the study.
Drugs and Chemicals
Purified recombinant apo A-IV and apo A-I were prepared as previously described (12). Sulfated CCK-8 (Sigma, St. Louis, MO) was dissolved in distilled water to make a 100-pmol/μl stock solution that was stored at −20°C and diluted immediately before use with 0.9% NaCl for motility experiments and with rat buffered saline (in mM: 140 NaCl, 5 KCl, 1 MgCl2·6H2O, 1.3 Na2HPO4, 5 HEPES, 2 CaCl2·2H2O, and 10 d-glucose; pH 7.38 ± 0.02) for electrophysiological experiments (5). The CCK1 receptor antagonist devazepide (Merck Sharp & Dohme, Rahway, NJ) was prepared by dissolving 10 mg in 0.1 ml DMSO (Sigma) and adding 0.1 ml Tween 80 (Sigma), followed by 0.8 ml physiological saline; this stock solution was diluted in physiological saline to achieve a final concentration of 1 mg/ml. Capsaicin (8-methyl-N-vanillyl-6-nonenamide, 10 mg; Sigma) was sonicated in 100 μl Tween 80 (Sigma) for 15 min, 0.9 ml olive oil was added, and the suspension was sonicated for a further 10 min.
Perineural Application of Capsaicin on the Vagus Nerve
This method has been published previously (20); briefly, rats were anesthetized with pentobarbital sodium (60 mg/kg ip). The carotid arteries were exposed by a midline neck incision, and the vagus nerve was carefully separated from the carotid arteries for a distance of 3–4 mm. A small stripe of parafilm (American National Can, Chicago, IL) was placed under the nerve, the nerve was wrapped with a small piece of cotton wool, and the surrounding tissue was covered with parafilm to prevent spread of capsaicin. One drop of 1% capsaicin or vehicle (10% Tween 80 in olive oil) was applied on each vagus nerve for 30 min. At 10-min intervals, the nerve was swabbed, and capsaicin was reapplied. After application, the area was rinsed thoroughly with sterile saline, and the incision was closed. Rats in which the vagus nerve was treated with vehicle (10% Tween 80 in olive oil) served as controls for the capsaicin treatment (intact rats). Animals were used 10 days after treatment.
Measurement of Gastric Motility
Methods have been described previously (20). Briefly, rats were anesthetized with urethane (1.25 g/kg ip; Sigma), and a catheter was placed in the trachea to ensure a clear airway (PE-240, 1.67 mm ID, 2.42 mm OD). The abdomen was opened, the pylorus was secured, gastric contents were gently flushed with warm 0.9% saline through an incision in the forestomach, and a catheter [2 mm inner diameter (ID), 3.2 mm outer diameter (OD); Silastic] was placed through the incision to measure intraluminal gastric pressure (IGP). After a recovery period of ∼45–60 min, the stomach was filled with 1 ml warm 0.9% saline and kept under continuous pressure of 5–6 cmH2O for 60 min to normalize baseline IGP. A catheter was placed in the femoral artery (PE-50, 0.58 mm ID, 0.965 mm OD) and advanced up to the junction with the celiac artery for close-arterial injection of apolipoproteins and CCK to the upper gastrointestinal tract. A second catheter was placed in the jugular vein for injections of drugs. IGP was displayed and recorded on-line for the duration of the experiment. Changes of IGP were measured and analyzed as the maximal decrease of IGP (cmH2O) in the postinjection period.
Recording of Vagal Afferent Nerve Fiber Discharge
The technique has been published previously (5). Briefly, rats were anesthetized and decapitated, and a segment of the thoracic esophagus, stomach, and proximal duodenum (∼4 cm from the pylorus to the common bile duct) was removed and immersed in oxygenated rat-buffered saline containing 2 g/l d-glucose. The esophagus and stomach were removed. With the use of a dissecting microscope, the subdiaphragmatic dorsal vagus nerve was identified, and a catheter was placed in the gastroduodenal artery for injections. The segment was pinned to the main chamber of a Sylgard-coated organ bath that was perfused continuously with oxygenated Ringer solution at a 2.0- to 2.5-ml/min flow rate, and the temperature of the organ bath was maintained at 33 ± 1°C. The isolated dorsal vagus nerve was placed in the recording chamber.
A thin nerve strand was isolated from the dorsal gastric vagus nerve trunk; the distal cut end was wrapped around one lead of a bipolar platinum-recording electrode, and a strip of neighboring connective tissue was wrapped around the other lead serving as the indifferent electrode. Action potentials of the afferent fibers were sent to a preamplifier (DAM-6 X100, 100- to 10-kHz bandpass filter; World Precision Instruments, Sarasota, FL), displayed on a digital storage oscilloscope (model 2211; Tektronix), and recorded on-line on a digital tape recorder (Sony high-density linear A/D D/A optical digital audio tape deck, DTC-700). Action potentials were sent simultaneously to a personal computer equipped with an A/D board (DT2831; Data Translation, Marlboro, MA).
Data Acquisition and Analysis
Single-unit activity of vagal afferents was discriminated from multiunit recordings using SPIKE 2 software (Cambridge Instruments, Cambridge, UK). Units within upper and lower threshold settings were acquired in the personal computer. On the basis of the amplitude and waveform, a particular unit can be matched to the waveform of the single unit by the use of the analysis module of the software in the off-line mode. The response pattern of different units can be analyzed further and displayed separately. Response magnitudes were normalized by a quotient (Q), where Q = 5-min spikes count of before/after the treatment. The Q >1.25 indicates an excitatory effect, Q <1.25 indicates an inhibitory effect, and Q = 1 ± 0.25 indicates no effect. Data are presented as means ± SE. Values were compared using Student's t-test (paired or unpaired) and were considered significantly different at P < 0.05.
Gastric motility experiments.
Gastric motility was measured in untreated control rats in response to administration of vehicle (physiological saline, 1 ml, n = 10), apo A-IV (200 μg in 1 ml, n = 10), apo A-I (200 μg in 1 ml, n = 10), and CCK (10 pmol in 0.1 ml). In perivagal capsaicin-treated rats (n = 7) or intact rats (n = 4), inhibition of gastric motility was measured in response to administration of apo A-IV (200 μg) and CCK (0.1, 1, and 10 pmol in 0.1 ml/rat). In rats treated with the CCK1 receptor antagonist devazepide (100 μg/kg iv, n = 4) or vehicle [0.1 ml DMSO, 0.1 ml Tween 80 (Sigma), and 0.8 ml physiological saline, n = 4], inhibition of gastric motility was measured in response to apo A-IV (200 μg) and CCK (0.1, 1, and 10 pmol). CCK was injected 15–60 min before and 15–60 min after devazepide treatment or in capsaicin-treated rats to ensure the effectiveness of CCK1 receptor blockade and vagal deafferentation, respectively.
In vitro electrophysiology.
Recordings were made from five preparations investigating the effects of apo A-IV on duodenal vagal afferent fiber discharge; a total of 10 single units were analyzed from these preparations. A dose-response to CCK was obtained (0.1, 1, 10, and 100 pmol) followed by apo A-IV (200 μg). At least 15 min were allowed between each injection.
In a further seven preparations, the effect of devazepide (100 μg intra-arterially) on the response to CCK (10 pmol) and apo A-IV (200 μg) was determined on vagal afferent fiber discharge. A total of 11 single units responding to CCK were analyzed from these preparations. After CCK injection, devazepide was administered, and injections of CCK and apo A-IV were repeated after 10–20 min.
Data are presented as means ± SE. Differences between groups were determined by a one-way ANOVA, followed by Student's t-test, using the software package of JMP (version 3.2.2; SAS Institute, Cary, NC). A probability of P < 0.05 was taken as significant.
Effect of Apo A-IV on Gastric Motility
As previously described (4), administration of apo A-IV (200 μg) was a potent stimulus to induce inhibition of gastric motility in anesthetized rats (Fig. 1). In contrast, close-arterial administration of the structurally similar apolipoprotein apo A-I or equivalent volumes of physiological saline (1 ml) had no effect on gastric motor function. In the same preparations, CCK (0.1, 1, and 10 pmol) induced a dose-dependent inhibition of gastric motor function (0.1 pmol CCK, 0.30 ± 0.12; 1 pmol CCK, 0.85 ± 0.19; 10 pmol CCK, 1.13 ± 0.18).
Effect of Functional Vagal Deafferentation on Apo A-IV-Induced Inhibition of Gastric Motility
In rats in which the vagus nerve was treated with capsaicin to produce a functional vagal deafferentation, apo A-IV (200 μg) was significantly less potent to inhibit gastric motility; theresponse was decreased by 55% compared with the response in intact rats (Fig. 2). The ability of CCK (0.1, 1, and 10 pmol) to inhibit gastric motility was reduced significantly in vagal capsaicin-treated rats compared with intact rats (decrease IGP in cmH2O after 0.1, 1, and 10 pmol ia CCK, intact vs. capsaicin treatments; 0.1 pmol CCK, 0.30 ± 0.12 vs. 0.04 ± 0.04; 1 pmol CCK, 0.85 ± 0.19 vs. 0.11 ± 0.07; 10 pmol CCK, 1.13 ± 0.18 vs. 0.63 ± 0.15; P < 0.05 for all CCK doses). Equivalent volumes of physiological saline had no significant effect on gastric motility in intact or capsaicin-treated rats.
Effect of CCK1 Receptor Blockade on Apo A-IV-Induced Inhibition of Gastric Motility
CCK1 receptor blockade using devazepide (100 μg/kg ia) markedly reduced the inhibition of gastric motility in response to apo A-IV by 77% (Fig. 2 ). Equivalent volumes of physiological saline had no significant effect on gastric motility in vehicle- or devazepide-treated rats. To demonstrate the effectiveness of the CCK1 receptor blockade, inhibition of gastric motility in response to CCK before and after devazepide treatment was reduced significantly (decrease of IGP in cmH2O after CCK, 10 pmol ia, before vs. after devazepide treatment; 0.98 ± 0.17 vs. 0.10 ± 0.06, 90% reduction, P < 0.01).
Effect of Apo A-IV on Duodenal Vagal Afferent Fiber Discharge
CCK produced a dose-dependent stimulation of duodenal afferent fiber discharge in 8 out of 10 units; the remaining 2 units did not respond to CCK (response Q for CCK-responsive units; vehicle: 1.07 + 0.05; 0.1, 1, 10, and 100 pmol CCK: 1.17 ± 0.18, 1.33 ± 0.13, 1.99 ± 0.38, and 2.35 ± 0.30, respectively). Apo A-IV stimulated the discharge of four of seven CCK-responsive units (response Q for apo A-IV-responsive units; 1.63 ± 0.21; P < 0.05, vehicle vs. apo A-IV; Fig. 3).
Duodenal vagal afferent fiber discharge in response to apo A-IV was reduced significantly by administration of the CCK1 receptor antagonist devazepide (response Q apo A-IV 1.75 ± 0.18 vs. 0.79 ± 0.10; P < 0.05, before vs. after devazepide treatment).
The data from the present study strongly suggest that apo A-IV participates in the sensory transduction pathway involved in the initiation of vagal afferent fiber activity and the consequent reflex decrease in gastric motility in response to luminal triglyceride. Our data show that apo A-IV stimulates vagal afferent fibers innervating the duodenum via a CCK1-responsive mechanism that results in a vagal reflex decrease in gastric motility. We postulate that active lipid absorption results in release of apo A-IV from enterocytes in the lamina propria to stimulate release of CCK from enteroendocrine cells in the intestinal epithelium. CCK stimulates vagal afferent fiber discharge via direct interaction with CCK1 receptors on vagal afferent terminals. In this way, the presence of lipid present in the intestinal lumen is signaled to vagal afferents and the central nervous system, resulting in changes in gastrointestinal function and possibly food intake.
These findings agree well with other evidence pointing to a role for chylomicron products, including apo A-IV, in mediating the changes in gastrointestinal function in response to long-chain triglyceride in the intestinal lumen. It is well recognized that intestinal infusion of triglyceride with fatty acids of chain length of at least C-12 (those that require chylomicron formation for absorption) and above induces release of CCK (9, 13) and inhibition of gastric motor function (8), but the mechanism by which these changes in function occur in response to lipid is not clear. The pathway involves CCK1 receptors, likely those located on vagal afferent nerve terminals in the intestinal mucosa, and a vago-vagal reflex pathway resulting in inhibition of gastric motor function and gastric emptying (7). Long-chain triglyceride increases vagal afferent fiber discharge, probably via an indirect, rather than direct, mechanism, since it is abolished in the presence of a CCK1 receptor antagonist (11). In addition, it has been shown to be dependent on chylomicron formation (18). Infusion of oleic acid stimulates intestinal vagal afferent fiber discharge, and this response is abolished when the lipid is infused with Pluronic L81, a hydrophobic nonionic surfactant that inhibits chylomicron formation (18). The results from the present study suggest that this may be because of the inhibition of apo A-IV secretion that occurs when chylomicron formation is blocked by Pluronic L-81.
The mechanism by which long-chain triglyceride stimulates the release of CCK from enteroendocrine cells is not known. Whether luminal fatty acids interact directly with these cells to cause secretion is unclear but has been the focus of several recent studies that have used the mouse neuroendocrine tumor cell line STC1. These cells secrete CCK in response to direct application of a number of different agents, including long-chain triglyceride (13, 22). Fatty acids of chain length C-10 and C-12 were effective in releasing CCK in a dose-dependent manner, and secretion was associated with an increase in intracellular calcium. However, it is not clear how these results with STC1 cells in vitro relate to enteroendocrine cells in situ. Caution must be used when extrapolating the results from tumor cell lines to native enteroendocrine cells; it is not known whether these cells express the same complement of proteins on the cell membrane or within the cell. In addition, in cell culture, the applied fatty acids have access to the whole cell membrane, whereas under physiological conditions, fatty acids have access to the luminal surface of the cell. Therefore, although these studies with STC1 cells suggest a direct effect of long-chain fatty acid on endocrine cells, it does not rule out the possibility of other pathways and mechanisms being involved under more physiological situations. Furthermore, it remains to be determined whether fatty acids stimulate apo A-IV secretion in STC1 cells and whether CCK is released from STC1 cells in response to apo A-IV. Previously, we have shown in awake rats that elevated plasma levels of CCK in response to luminal triglyceride perfusion are reduced significantly by simultaneous perfusion with Pluronic L81 (19). This result suggests that chylomicron formation is an important step in the release of CCK from intestinal EC cells. The sequence of events leading to fatty acid-induced release of CCK from EC cells is not clear; it is possible that apo A-IV released from chylomicrons in the interstitium activates EC cells that release CCK. An alternate hypothesis is that endocrine cells might synthesize the components required for chylomicron formation and thus produce apo A-IV, which could then have an autocrine effect to stimulate release of CCK. This is currently unknown; although immunocytochemistry with an antibody to apo A-IV showed immunoreactivity in the intestinal epithelial cells (1), the colocalization of apo A-IV with CCK has not been studied. In addition, the possibility of absorption of fatty acids in endocrine cells has not been demonstrated in vivo, although it has been shown that STC1 cells absorb dodecanoic acid in cell culture (22). Whether endocrine cells express any of the normal proteins involved in handling of free fatty acids by cells, such as fatty acid transporters and binding proteins, is not known.
Regardless of the mechanism by which endocrine cells release CCK, it is clear that apo A-IV can stimulate vagal afferent fiber discharge via a CCK1-dependent mechanism. The simplest explanation is that apo A-IV stimulates CCK release and then CCK binds to CCK1 receptors on vagal afferent terminals. The observation that CCK directly stimulates vagal afferents has been shown in a number of different experimental paradigms. Furthermore, vagal afferents have been shown to express CCK1 receptors, although localization of the receptor to the terminal fields of intestinal vagal afferents has never been demonstrated directly. However, this does not rule out the possibility that apo A-IV may have a direct effect on vagal afferent nerve terminals. Apo A-IV has been shown to have effects in the central nervous system, presumably mediated by a direct effect on neurons or possibly on glia (3, 14–16). It is not clear whether apo A-IV exerts its biological actions via specific receptors. In addition, the lack of response to apo A-IV may reflect a general decrease in sensitivity of vagal afferents to any stimuli in the presence of CCK1 receptor blockade. Further information is required on the direct effects of apo A-IV on endocrine cells and vagal afferents to help clarify this pathway.
In conclusion, we have shown that apo A-IV activates a vagal afferent, CCK1 receptor-dependent pathway to inhibit gastric motility in rats. The significance of this finding is that it provides further insights into the mechanism by which fatty acids from triglyceride are sensed in the intestinal epithelium to produce alterations in postprandial gastrointestinal function and regulation of food intake. The precise mechanism by which apo A-IV stimulates release of CCK and activates vagal afferents warrants further investigation.
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-41004 (H. E. Raybould) and DK-52149, DK-56863, and DK-54504 (P. Tso) and Deutsche Forschungsgemeinschaft Grant GL 311/ 3-1.
We are very grateful to Jen Yu Wei, David Adelson, and Yu Hua Wang (CURE Digestive Diseases Research Center, University of California Los Angeles School of Medicine) for help with the electrophysiological experiments.
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