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APPETITE, OBESITY AND METABOLISM
1Department of Anatomy, Physiology, and Cell Biology, School of Veterinary Medicine, University of California, Davis 95616; 3Department of Biology, California State University Sacramento, Sacramento, California 95819; 2University Hospital, Department of General Surgery, University of Tübingen, 72074 Tübingen, Germany; 4Department of Surgery, Louisiana University School of Medicine, Shreveport, Louisiana 71130; and 5Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, Ohio 45267
Submitted 9 December 2003 ; accepted in final form 13 April 2004
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ABSTRACT |
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lipid; chylomicrons; mesenteric lymph; cholecytokinin
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.
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METHODS |
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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.
Experimental Protocols
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.
Statistical Analysis
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.
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RESULTS |
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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).
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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.
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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).
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DISCUSSION |
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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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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