Lipid, particularly long-chain triglyceride, initiates feedback regulation of gastrointestinal function. To determine whether the site of action of lipid is pre- or postabsorptive, we investigated the ability of mesenteric lipid-fed lymph to inhibit gastric motor function. Lymph was collected from awake lymph-fistula rats during intestinal infusion with either a glucose-saline maintenance solution or lipid. Intra-arterial injection of lymph collected during intestinal lipid infusion significantly inhibited gastric motility in anesthetized recipient rats compared with injection of equivalent amounts of triglyceride or lymph collected during intestinal infusion of maintenance solution. Lymph collected from rats during lipid infusion with Pluronic L-81 [an inhibitor of chylomicron formation and apolipoprotein (apo) A-IV secretion] compared with lymph injection from donor animals treated with Pluronic L-63 (a noninhibitory control for Pluronic L-81) was significantly less potent. Injection of purified recombinant apo A-IV significantly inhibited gastric motility. Products of lipid digestion and absorption, other than fatty acids or triglyceride, released by the intestine during lipid digestion likely serve as signals to initiate intestinal feedback regulation of gastrointestinal function. Most likely, apo A-IV is one of the signals involved.
- apolipoprotein A-IV
- long-chain triglyceride
- Pluronic L-81
digestion and absorption of nutrients in the intestine elicit feedback regulation of proximal gastrointestinal (GI) function, such as inhibition of gastric emptying (22) and acid secretion (17), alteration of intestinal motility (2), and alteration in behaviors such as food intake and satiety (33). For different macronutrients, different neuronal and humoral pathways mediate these responses. For example, inhibition of gastric emptying and gastric acid secretion in response to intestinal lipid is mediated by a vagal capsaicin-sensitive and CCKAreceptor-mediated pathway (11, 17). Lipid in the small intestine stimulates the release of CCK from endocrine cells (11,13, 19), and there is evidence that postprandially released CCK activates CCKA receptors on vagal afferent nerve terminals in the mucosa (11, 17). This in turn stimulates neurons in the nucleus of the solitary tract, a brain area involved in regulation of proximal GI function and food intake (20, 33).
It is well recognized that intestinal infusion of fatty acids of chain length greater than C12 induce a significant inhibition of gastric motility (12) and that long-chain triglycerides (TGs) release CCK (13). It has recently been shown that there is a good correlation between increase in plasma levels of CCK and inhibition of gastric motility with fatty acids of chain length above but not below C10 in humans (19). Increased apolipoprotein (apo) A-IV output into the mesenteric lymph is observed in response to long-chain fatty acids, i.e., C14–C20 (14-16), and plasma levels of apo A-IV increase rapidly after lipid administration (24). It has been proposed that apo A-IV is involved in postprandial regulation of food intake. Peripheral administration of apo A-IV has been shown to inhibit food intake and gastric emptying (4, 27). Increased levels of apo A-IV were measured in the cerebrospinal fluid after lipid ingestion, and reduced food intake was also observed following intracerebrovascular injection of apo A-IV (5).
We have previously shown that inhibition of gastric emptying in response to lipid is dependent on the formation of chylomicrons, since blockade of chylomicron formation using Pluronic L-81 prevented the ability of lipid in the intestine to inhibit gastric emptying (23). However, it is not clear if it is the TG content of the chylomicrons or some other component that is signaling to either the endocrine cells or extrinsic primary afferent neurons. In the present study, we tested the hypothesis that inhibition of gastric motor function is dependent on the postabsorptive components of intestinal lipid digestion and absorption, possibly chylomicrons and/or chylomicron components, for example apo A-IV, rather than the products of lipid absorption (TG) itself. The effect of postabsorptive chylomicron products was studied by measuring the ability of chylous lymph given peripherally to inhibit gastric motility in recipient rats.
Male Sprague-Dawley rats (200–250 and 300–350 g; Harlan Industries, San Diego, CA) maintained on regular laboratory chow were housed under controlled conditions of illumination (12:12-h light/dark cycle starting at 7:00 PM), 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.
Mesenteric Lymph Preparation
Methods were published previously (16). Briefly, rats (300–350 g) were anesthetized with methohexital sodium (60 mg/kg ip; Brevital, Jones Pharma, St. Louis, MO). The mesenteric lymph duct was cannulated with a polyvinyl chloride tube (medical grade; 0.50 mm ID, 0.80 mm OD; Dural Plastics and Engineering, Dural, NSW, Australia), fixed in place with a drop of ethyl cyanoacrylate glue (Krazy Glue, Elmer's Products, Columbus, OH), and externalized through a stab wound in the right flank. A second cannula (Silastic; 1 mm ID, 2.15 mm OD) was passed through the fundus of the stomach, extended 3 cm into the duodenum, secured in place with a silk suture, and additionally fixed with a drop of ethyl cyanoacrylate glue. After surgery, rats were placed in Bollman cages and allowed to recover, and a glucose-saline solution (0.2 mol/l glucose, 145 mmol/l NaCl, and 4 mmol/l KCl) was perfused continuously through the duodenal cannula at a rate of 3 ml/h for the 24-h recovery period. The purpose of this maintenance solution was to ensure adequate hydration and nutritional status of the animals.
Determination of TG and glucose in lymph.
Glucose levels of abdominal lymph were measured by a glucose-oxidase test and expressed as milligrams per 100 milliliters. TG content of the mesenteric lymph was determined by a quantitative enzymatic measurement with TG ultraviolet reagents (TG kit A-334, Sigma, St. Louis, MO) and analyzed at 340 nm (1).
Measurement of Gastric Motility
Rats (200–250 g) were anesthetized with urethane (1.25 g/kg ip; Sigma), and a catheter was placed into the trachea to ensure a clear airway (polyethylene tubing, PE-240; 1.67 mm ID, 2.42 mm OD) (10). The abdomen was opened, the pylorus was secured, gastric contents were gently flushed with warm saline (0.9%) through an incision in the forestomach, and a catheter (Silastic; 2 mm ID, 3.2 mm OD) was placed through the incision to measure intraluminal gastric pressure (IGP). After a recovery period of ∼45–60 min, the stomach was filled with warm saline (0.9%, 1 ml) and kept under continuous pressure of 5–6 cmH2O for 60 min to normalize baseline IGP. Catheters were placed in the jugular vein and femoral artery (PE-50; 0.58 mm ID, 0.965 mm OD) for administration of lymph or drugs. The catheter in the femoral artery was advanced up to the junction with the celiac artery for injections close-arterial to the upper GI tract. IGP was displayed and collected online for the duration of the experiment. Changes of IGP were measured and analyzed as the maximal decrease of IGP (cmH2O).
Collection of mesenteric lymph.
After the 24-h recovery period, abdominal lymph was collected for 2 h during infusion of the glucose-saline maintenance solution (0.2 mol/l glucose, 145 mmol/l NaCl, and 4 mmol/l KCl; 3 ml/h) and for 6 h during lipid-saline infusion (Intralipid 20%, Baxter, Deerfield, IL; TG infusion: ∼34 μmol/h, n = 3; ∼85 μmol TG/h, n = 3; ∼170 μmol/h,n = 16; ∼340 μmol/h, n = 3; in saline solution, 145 mmol/l NaCl 4 mmol/l KCl, at 3 ml/h). Lymph samples were collected in ice-chilled tubes for time intervals of 2 h and frozen at −85°C. Lymph was also collected from rats, infused with lipid (170 μmol/h TG) and either Pluronic L-81 (L-81; BASF, Wyandotte, MI; n = 15) or Pluronic L-63 (l-63; BASF; n = 12). L-81 or L-63 were dissolved in five drops of ethanol, then added to the lipid-saline solution (final concentration, 4 mg/h). L-81 is a hydrophobic surfactant that inhibits chylomicron formation and apo A-IV synthesis, whereas L-63, a structurally similar surfactant, has no such effect and served as a control (29-31).
Gastric motility experiments.
Gastric motility was measured in three groups of recipient animals. Only chylous lymph obtained from rats perfused with 170 μmol TG/h was used in gastric motility experiments. Lymph samples from a single donor rat were used for each experiment on recipient rats. Lymph samples were defrosted, stirred, centrifuged at 1,000 g to eliminate cells and clots, and injected either intravenously or intra-arterially. The first group of animals (n = 15) received injection with lymph (1 ml, iv and ia) from animals infused with either maintenance solution (control lymph) or lipid-saline solution (chylous lymph) or receiving control injections with equal amounts of glucose (175 mg/dl) or TG (Intralipid; ∼11.3 μmol TG/ml) contained in control or chylous lymph, respectively. Another group (n = 15) was injected with lymph (1 ml ia) from animals infused with lipid + L-63 or lipid + L-81. Finally, a group of rats (n = 10) was injected with purified apo A-IV (200 μg/ml ia), prepared as previously described (4), or vehicle (physiological saline solution, 1 ml). In each experiment, sulfated CCK-8 (10 pmol in 0.1 ml physiological saline ia; Sigma) was injected; this served as positive control to ensure the integrity of the preparation.
Data are presented as means ± SE. Differences between groups were determined by ANOVA, followed by Student'st-test using the JMP software package (version 3.2.2, SAS Institute, Cary, NC). A probability of P < 0.05 was considered significant.
Effect of Intestinal Glucose and Lipid Infusion on Mesenteric Lymph Flow and TG Output
After the 24-h recovery period, abdominal lymph flow during intestinal glucose-saline infusion reached a steady state of ∼2.6 ml/h in all groups (Table 1). Glucose concentration of this control lymph was 175 ± 7 mg/dl (9.7 ± 0.4 mmol/l; n = 10), representing a glucose output of 4.4 ± 0.4 mg/h (0.24 ± 0.02 mmol/l).
Administration of lipid (34–170 μmol/h TG) in the gut dose-dependently increased mesenteric lymph flow. A maximal flow rate of 3.3 ml/h was obtained with 170 μmol/h TG during the 5–6 h of lipid infusion. A further increase of the intestinal lipid dose to 340 μmol/h significantly decreased mesenteric lymph flow, and a fatty diarrhea was observed. There was no significant increase in mesenteric lymph flow during intestinal lipid infusion (170 μmol TG/h) with either L-81 or L-63.
Lipid infusion (34–170 μmol/h TG) significantly increased the TG concentration and TG output. TG output dose-dependently increased during intestinal lipid infusion (34–170 μmol/h TG). Maximum TG output was obtained with 170 μmol/h TG, producing a fivefold increase in both the 1st through 2nd hours and 5th through 6th hours of lipid infusion (Table 1). TG concentration increased, but total TG output decreased in rats infused with 340 μmol TG/h. In L-81 treated rats, TG concentration and TG output was significantly reduced compared with lipid perfusion alone or lipid perfusion with the control surfactant L-63 during the first 2 h of lipid infusion (TG concentration in μmol/ml: 7.86 ± 0.66 vs. 5.22 ± 0.57; TG output in μmol/h: 15.58 ± 1.47 vs. 10.14 ± 1.25; bothP < 0.05 for lipid + L-63 vs. lipid + L-81).
Effect of Mesenteric Lymph on Gastric Motility
Gastric motility was significantly inhibited by injection of lymph collected during intestinal lipid infusion into recipient rats (Fig.1). There was no significant difference in the inhibition of gastric motility in response to chylous lymph harvested at 1–2 h or 5–6 h of lipid infusion. Injection of mesenteric lymph taken during perfusion of maintenance solution (control lymph) also inhibited gastric motility, but this was significantly less than that obtained following injection of chylous lymph (decrease IGP: 0.75 ± 0.10 vs. 0.44 ± 0.09 cmH2O; P < 0.05 control lymph vs. 1–2 h chylous lymph). An isovolumic control injection containing identical amounts of either glucose or TG had a small effect on gastric motility that was significantly less than the inhibition in response to either control or chylous lymph (P < 0.01; Fig.2).
The route of injection of either control or chylous lymph had no significant effect on the magnitude of the response (decrease of IGP in cmH2O: chylous lymph, 0.65 ± 0.19 vs. 0.75 ± 0.1, iv vs. ia; control lymph, 0.51 ± 0.18 vs. 0.44 ± 0.09, iv vs ia; not significant).
Effect of Mesenteric Lymph Following Perfusion of Lipid With L-81 or L-63 on Gastric Motility
Inhibition of gastric motility in response to injection of mesenteric lymph harvested from rats perfused with lipid containing L-81 was significantly reduced compared with lymph from rats perfused with lipid + L-63 or chylous lymph, representing a decrease of ∼40–45% compared with chylous lymph or lymph from L-63-treated rats (Fig. 3).
Effect of Apo A-IV on gastric motility.
Injection of purified apo A-IV (200 μg/ml ia), approximately equivalent to that contained in 1 ml of chylous lymph, significantly inhibited gastric motility compared with injection of either vehicle injection (0.9% NaCl, 1 ml) or recombinant purified apo A-I (200 μg/ml), which has a molecular structure and weight comparable with apo A-IV (Fig. 4).
Effect of CCK on Gastric Motility
CCK produced a significant and consistent decrease in GI motility in following either intra-arterial or intravenous injection (decrease IGP in cmH2O: 1.25 ± 0.18 vs. 1.13 ± 0.16, ia vs. iv; not significant).
The present study demonstrates that chylous lymph significantly inhibits gastric motility; this effect was not dependent on the lipid content of the chylous lymph but was dependent on the presence of chylomicrons. Additionally, injection of apo A-IV, a component of chylomicrons, inhibited gastric motility. This was unlikely to be due to a nonspecific effect since injection of apo A-I, another gut peptide associated with chylomicrons that shares some functions with apo A-IV, except inhibition of food intake (4), had no effect. These data suggest that a component of chylomicrons, possibly apo A-IV, is involved in the activation of the CCKA receptor and vagal afferent pathway mediating lipid-induced inhibition of gastric motor function. It is possible that apo A-IV may stimulate the release of CCK; CCK would then bind to CCKA receptors on vagal afferent nerve terminals to stimulate a vago-vagal decrease in gastric motility. It is well established that intestinal lipid inhibits gastric motor and secretory function by a CCKA and vagal afferent pathway (11, 17). It has recently been shown that intestinal lipid absorption stimulates the discharge of vagal afferent fibers innervating both the ileum and jejunum and that this effect is dependent on formation of chylomicrons since it was blocked by concomitant administration of L-81 (22). Together with the present data, in particular the effectiveness of apo A-IV but not TG to reproduce this inhibition of gastric motility, provides strong evidence for not only a postabsorptive site of action of lipids to activate vagal afferents and influence gastric motility but also that this is dependent on chylomicrons, possibly apo A-IV.
Initial experiments determined the effect of different doses of Intralipid on mesenteric lymph flow or output; this enabled us to select a dose of lipid that produced a significant, reliable, and sustained TG output. Chylous lymph was collected during intestinal Intralipid infusion, a lipid emulsion containing only long-chain TGs with a chain length of C18 (89.2%) or C16 (10.8%) (26). Since maximal stimulation of mesenteric lymph flow and TG output was observed with a dose of 170 μmol TG/h and diarrhea was absent, this dose was used to harvest chylous lymph in donor experiments.
The hydrophobic surfactant L-81, infused intestinally with the lipid, significantly inhibits chylomicron formation and thus the appearance of TG in mesenteric lymph (7, 21). In L-81-treated animals, the absorbed lipid accumulates in the intestinal mucosa and is not transported into the lymph (29-31). In the present study, a significant decrease of TG concentration and TG output was also observed in L-81-treated rats compared with untreated control rats or L-63-treated rats, consistent with these other studies. Importantly, injection of lymph from L-81-treated rats was significantly less potent to inhibit gastric motility compared with either chylous lymph or lymph from L-63-treated rats. Thus there was a consistent motility response to chylous lymph taken from untreated rats or from L-63-treated rats compared with lymph taken during blockade of chylomicron formation and secretion. Additionally, control injections containing equivalent amounts of lipid did not inhibit gastric motility. Intralipid emulsion has often been used for studying chylomicron metabolism; it contains chylomicron-like emulsion particles, made of a triacylglycerol core emulsified by a monolayer of phospholipids, but does not contain apoproteins or cholesterol (25). Together, these results suggest that postabsorptive products like chylomicrons or chylomicron products are involved in the negative feedback regulation of gastric motor function. It has been shown that L-81 also reduced the appearance of apo A-IV in mesenteric lymph (7), consistent with its effects on chylomicron formation. Since apo A-IV inhibited gastric motility, it is possible that the active constituent of chylomicrons is apo A-IV.
It is interesting that lymph collected during perfusion of the glucose/electrolyte maintenance solution also inhibited gastric motility. It is likely that this lymph contains additional factors such as cytokines or catecholamines, since lymph samples are collected in a postoperative status. Part of the inhibitory effect on gastric motility of control or chylous lymph therefore could be cytokine related, since tumor necrosis factor, a proinflammatory cytokine, is able to inhibit gastric motility under certain conditions (8, 9). However, the mechanism by which control lymph inhibits gastric motility was not perused further in the present study.
The major products of intraluminal lipid digestion are 2-monoglyceride and fatty acids. Digestion products with chain length greater than C10 are absorbed by the enterocytes and migrate from the site of uptake to the endoplasmic reticulum, where they are resynthesized into TG, packed into chylomicrons, removed from the enterocyte by exocytosis, and transported via the mesenteric lymph (18, 28). Fatty acids of chain length less than C10 diffuse out of enterocytes and are primarily transported in the portal vein to the liver, bound to albumin (28). Apo A-IV in rats is mainly synthesized in the intestine, stimulated by long-chain fatty acids, which are transported via the lymph on chylomicrons, but a minor amount synthesized in the liver is released independently of intestinal nutrients (15,32). In the intestinal lymph, TG-rich lipoproteins such as chylomicrons and very-low-density lipoproteins contain one-half of the total amount of apo A-IV (6). When the TG-rich lipoproteins enter the circulation, apo A-IV is displaced from chylomicrons to high-density lipoproteins and lipoprotein-free fraction (3). After entering the circulation, a small amount of apo A-IV enters the nervous system, and there is evidence that apo A-IV is involved in central inhibition of food consumption, since apo A-IV injected centrally at a dose of only 0.5 μg is able to inhibit food intake significantly (5). A similar pathway for the apo A-IV-induced inhibition of gastric motility could be possible; however, peripheral activation of vagal afferent neurons is also conceivable. These data suggest that a component of chylous lymph associated with chylomicron formation, probably apo A-IV, is involved in the feedback regulation of gastric motility.
In conclusion, the present studies have demonstrated that it is a component of chylous lymph that initiates the decrease in gastric motility in response to lipid in the intestine. Moreover, a component of chylomicrons, apo A-IV, also inhibits gastric motility and may be the active constituent of chylous lymph. Since the effect of lipid to inhibit gastric motility is mediated by CCK acting at type A receptors located on vagal primary afferent nerve terminals, it is possible that apo A-IV may be involved in sensory transduction in the intestine.
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-41004 (to H. E. Raybould), DK-52149, DK-56910, DK-56863, and DK-54504 (to P. Tso) and by Deutsche Forschungsgemeinschaft Grant GL 311/1–1 (to J. Glatzle).
Address for reprint requests and other correspondence: H. E. Raybould, 1321 Haring Hall, Univ. of California, Davis, CA 95616 (E-mail:).
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