Stimulation of jejunal synthesis of apolipoprotein A-IV by ileal lipid infusion is blocked by vagotomy

Theodore J. Kalogeris1, V. Roger Holden1, and Patrick Tso2

1 Department of Surgery, Louisiana State University Medical Center, Shreveport, Louisiana 71130; and 2 Department of Pathology, University of Cincinnati, Cincinnati, Ohio 45267


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We examined the role of vagal innervation in lipid-stimulated increases in expression and synthesis of intestinal apolipoprotein A-IV (apoA-IV). In rats with duodenal cannulas and superior mesenteric lymph fistulas given duodenal infusions of lipid emulsion, vagotomy had no effect on either intestinal lipid transport, lymphatic apoA-IV output, or jejunal mucosal apoA-IV synthesis. In rats with jejunal Thiry-Vella fistulas, ileal lipid infusion elicited a twofold stimulation of apoA-IV synthesis without affecting apoA-IV mRNA levels; vagotomy blocked this increase in apoA-IV synthesis. Direct perfusion of jejunal Thiry-Vella fistulas produced 2- to 2.5-fold increases in both apoA-IV synthesis and mRNA levels in the Thiry-Vella segment; these effects were not influenced by vagal denervation. These results suggest two mechanisms whereby lipid stimulates intestinal apoA-IV production: 1) a vagal-dependent stimulation of jejunal apoA-IV synthesis by distal gut lipid that is independent of changes in apoA-IV mRNA levels and 2) a direct stimulatory effect of proximal gut lipid on both synthesis and mRNA levels of jejunal apoA-IV that is independent of vagal innervation.

ileal brake; nutrient absorption; chylomicrons; Thiry-Vella fistula; lymph fistula; triglyceride


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

APOLIPOPROTEIN (apo) A-IV is synthesized in intestinal mucosa and secreted into intestinal lymph during absorption and transport of a lipid meal (12, 13, 16). The physiological function of apoA-IV is under active investigation. Several studies have provided evidence that apoA-IV may play a role in cholesterol and lipoprotein metabolism (7, 10); more recently, strong in vivo evidence supports roles for apoA-IV in the control of ingestive behavior (9) and gastric function (23, 24) and in protection against lipoprotein oxidation (30) and atherosclerosis (3, 6). Because the intestine contributes the major proportion of circulating apoA-IV, it is important to understand the physiological mechanisms involved in the control of apoA-IV expression and release. These mechanisms are not clearly understood.

It is well established that intestinal apoA-IV synthesis and secretion are stimulated by dietary fat. Increased secretion of apoA-IV into intestinal lymph (13, 16) and increases in plasma levels of apoA-IV (33) have been demonstrated in response to single lipid meals or chronic intake of a high-fat diet. Graded doses of duodenally infused triacylglycerol produced graded increases in both lymphatic apoA-IV output and in intestinal mucosal synthesis, along a proximal-to-distal gradient (16). Available evidence indicates that increases in apoA-IV synthesis can be explained by a pretranslational mechanism (1, 11, 21). Other studies have provided evidence that stimulation of intestinal apoA-IV synthesis and secretion depend on assembly and transport of chylomicrons (13). Thus apoA-IV synthesis and secretion are stimulated by some aspect of intestinal lipid transport. However, the mechanism whereby dietary lipid stimulates intestinal apoA-IV is virtually unknown.

Recently, we (21) obtained evidence that intestinal apoA-IV synthesis may be controlled, at least in part, by an indirect mechanism. In response to duodenal infusions of lipid emulsion, apoA-IV mRNA levels and biosynthesis in the proximal jejunum were significantly increased, whereas ileal apoA-IV synthesis and message levels remained unaffected. However, ileal lipid infusions stimulated synthesis of apoA-IV in both ileum and proximal jejunum. Moreover, ileal but not duodenal lipid infusion also stimulated apoA-IV synthesis in jejunal Thiry-Vella fistulas, indicating that the effect of distally infused lipid on proximal intestinal synthesis of apoA-IV was independent of the presence of jejunal lipid. Finally, perfusion of ileal Thiry-Vella fistulas was sufficient to significantly stimulate proximal jejunal apoA-IV synthesis. These findings suggest the existence of a signal, elicited by lipid in the distal gut, capable of increasing synthesis of apoA-IV in other regions of the intestine. Moreover, in view of recent findings by Okumura et al. (23, 24) that apoA-IV has enterogastrone-like actions on the stomach, the above findings further suggest that stimulation of apoA-IV by lipid in the distal gut may be a component of the "ileal brake" (19, 21, 31).

The nature of the putative ileal signal is unknown. Because the Thiry-Vella fistulas used in the above studies had an intact blood supply and retained extrinsic innervation, either a neural or hormonal or a combined neurohumoral signaling mechanism might be involved. However, any attempt to address the role of a putative mediator of the dietary lipid effect on intestinal apoA-IV must also define the effect (if any) of that mediator on the process of lipid absorption and transport, which until recently (20) was thought to be the only consistent, documented stimulus for intestinal apoA-IV (19). The possible role of neural/hormonal signals in the absorption and transport of dietary fat by the intestine has not been previously examined.

Stimulation of jejunal apoA-IV synthesis by duodenal lipid infusion has been shown to be accompanied by an increase in apoA-IV mRNA levels (21). However, it is not known whether this is also the case in response to ileal lipid infusion; i.e., it is not known whether there is only one mechanism underlying increases in intestinal apoA-IV production in response to lipids.

The purpose of the present study was to examine whether intact vagal innervation is required for 1) normal lipid absorption and transport, 2) the coupling of synthesis and secretion of apoA-IV to intestinal lipid transport, and 3) the comparative stimulatory effects of ileal vs. direct jejunal lipid infusion on proximal jejunal gene expression and synthesis of apoA-IV.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals and Surgical Preparation

Male Sprague-Dawley (Harlan, Indianapolis, IN) rats were used for all studies. They were maintained on a 12:12-h light-dark cycle with lights on at 0600. Except where noted, animals were allowed free access to food (Teklad Rodent Chow, Harlan) and water.

Total subdiaphragmatic vagotomy. Rats were deprived of food overnight and then anesthetized by intraperitoneal injection with pentobarbital sodium (55 mg/kg). They were laparotomized using a midline incision. The liver was retracted anteriorly using gauze pads moistened with physiological saline solution, and gentle traction was placed on the stomach. With the use of blunt dissection under a dissecting microscope, the anterior and posterior trunks of the vagus nerve were exposed and identified. Both trunks were carefully isolated from the surrounding fascia, adipose, and blood vessels and then sectioned using an electrocautery, using caution to not damage the surrounding tissue. The anterior trunk on the ventral aspect of the esophagus was cut rostrally to the bifurcation of the hepatic branch; the posterior trunk was cut rostrally to the posterior gastric and celiac branches. Control rats were subjected to sham surgery, in which the liver and stomach were retracted as above, and the vagal trunks were exposed but left intact. After closure of the incision, rats were allowed to recover fully from anesthesia in a cage with a heating pad. They were then returned to their home cages and allowed free access to water and a nutritionally complete liquid diet (ISOCAL, Mead Johnson, Evansville, IN). Use of the liquid diet allowed more rapid postoperative recovery in the face of the gastric stasis, which normally occurs after total vagal denervation. After 6-8 days, the rats were deprived of food for 24 h and then equipped with intestinal cannulas with or without intestinal lymph duct fistulas; intestinal infusion and lipid and apoA-IV transport or apoA-IV synthesis studies and brain fixation were performed the following day.

Verification of vagotomy using fluorogold retrograde tracer technique. Verification of the completeness of vagotomy was performed as previously described (28). Use of the retrograde tracer method for verification of vagotomy necessitated the administration of fluorogold to each rat 3 days before lipid infusion studies. Four to six days after sham surgery or vagotomy, each rat was given an intraperitoneal injection of fluorogold (0.5 mg/rat, Fluorochrome, Englewood, CO). Lipid infusion studies were carried out 3 days after fluorogold injection. At the end of each experiment, rats were perfused transcardially with 400 ml of warm saline (37°C) and then 800 ml of 10% phosphate-buffered formalin. Brains were removed and postfixed in 15% sucrose in formalin at 4°C. The medulla and brain stem were frozen in M-1 embedding matrix (Lipshaw, Pittsburgh, PA), and serial, 56-µm coronal sections between the level of the facial nucleus and the pyramids were collected onto gelatin-coated microscope slides and air dried overnight. Slides were treated with graded concentrations of ethanol (70, 95, and 100%) and xylene and then coverslipped with DPX mounting medium (Aldrich, Milwaukee, WI). After drying, slides were examined with a Zeiss epifluorescence system as described previously (28). Completeness of vagotomy was assessed on the basis of the difference in intensity of fluorogold staining between the dorsal motor nucleus of the vagus (DMX) and the hypoglossal nucleus. Rats not showing similarly diminished staining throughout the DMX, indicating complete destruction of both anterior and posterior branches, were judged to be incompletely vagotomized, and data from these rats were therefore eliminated from analysis. Animals found to have indeterminate vagotomies, i.e., in which no fluorogold labeling was seen in both the DMX and the area postrema (staining in the latter region being a positive quality control for proper fluorogold administration), were similarly eliminated from analysis.

Implantation of duodenal or ileal infusion cannulas and mesenteric lymph duct fistulas. One week after denervation or sham surgery, rats were deprived of food overnight and then anesthetized with halothane. For lymphatic apoA-IV and lipid transport studies, rats were equipped with duodenal and intestinal lymph duct cannulas. The superior mesenteric lymph duct was cannulated with vinyl tubing (0.8 mm OD; Dural Plastics and Engineering, Dural, New South Wales, Australia) as described previously (16). A silicone tube (1.6 mm OD) was passed through the forestomach, extended 1-2 cm into the duodenum, and then secured in place with a purse-string suture (4-0 silk) and a drop of cyanoacrylate glue. For apoA-IV synthesis studies, rats were equipped with either duodenal cannulas (as described above) or with ileal cannulas. For the latter, vinyl tubing (1.27 mm OD) was introduced into the ileum ~18-20 cm proximal to the ileocecal junction and secured with a purse-string suture (6-0 silk) and a drop of cyanoacrylate glue. Tubes were exteriorized through stab wounds in the animal's right flank, and then rats were placed in restraint cages (16) in a temperature-regulated chamber at 30°C and allowed to recover for 24 h. During recovery, rats with lymph fistulas received continuous duodenal infusions of a glucose-saline solution (145 mM NaCl, 4 mM KCl, and 0.28 M glucose) at a rate of 3 ml/h. Rats with duodenal or ileal cannulas only (i.e., without lymph fistulas) received continuous duodenal or ileal infusions of glucose-saline solution at 0.85 ml/h. Rationale for using this lower infusion rate for apoA-IV synthesis studies was previously described; briefly, the lower rate was necessary to prevent diarrhea in ileally infused rats (21).

Preparation of jejunal Thiry-Vella fistulas. Rats were prepared with Thiry-Vella fistulas on the same day as receiving either vagotomies or sham denervation. This surgery was modified from the previously described procedure (21). Briefly, a 20-cm segment of proximal jejunum was resected from the gut, taking care to preserve all attached mesentery containing its blood supply and extrinsic neural innervation. Its proximal end was sutured closed, and its distal end was exteriorized through stab wounds in the animal's right flank and sutured to the skin to form an jejunostomy. A vinyl catheter (0.86 mm ID, 1.27 mm OD, Dural) was inserted into the closed, proximal end of the Thiry-Vella segment and fixed in place with a purse-string suture and a drop of cyanoacrylate glue. Beginning 48 h after surgery, and for the duration of the recovery period, the Thiry-Vella segment was perfused daily with 1 ml of a solution of 5% glucose in saline containing 1 mM L-glutamine. After 6 days of recovery, rats were deprived of food for 24 h and then implanted with ileal cannulas as described above.

Lipid Infusates

Two types of lipid infusates were used. In the lipid and apoA-IV lymphatic transport studies, rats were infused with a lipid emulsion containing 40 µmol triolein (labeled with 1.2 µCi [3H]triolein), 8.7 µmol egg phosphatidylcholine, and 57 µmol sodium taurocholate and sonicated in 3 ml PBS, pH 6.4 (16). This emulsion was infused at 3 ml/h for 8 h. Rationale for this dose of triglyceride was previously discussed (16). For apoA-IV synthesis studies, rats were infused with lipid emulsions containing monoolein (20 µmol/h), oleic acid (40 µmol/h; equivalent to a triglyceride dose of 20 µmol/h), and sodium taurocholate (57 µmol/h) in PBS. This latter lipid dose was previously shown to produce an apoA-IV synthetic response similar to the response at the higher dose used for the apoA-IV transport studies (16, 21). For experiments, monoglyceride plus fatty acid emulsions were infused at 0.85 ml/h for 8 h.

Experimental Procedures

Experiment 1: Effect of vagotomy on lymphatic lipid transport and apoA-IV secretion during duodenal infusion of triacylglycerol. Two groups of rats were used, vagotomized rats (n = 5) and sham-vagotomized rats (n = 6). Animals were prepared with duodenal cannulas and intestinal lymph fistulas and allowed to recover for 24 h, during which time they received continuous duodenal infusion of glucose-saline. At the start of the experiment, lymph was collected for 1 h before lipid infusion (fasting lymph). The infusate was then switched to triolein emulsion containing [3H]triolein. From the start of lipid infusion, lymph was collected at 2, 4, 5, 6, 7, and 8 h. All lymph samples were collected in ice-chilled glass tubes. Aliquots of lymph were taken for measurement of radioactivity and for assay of apoA-IV. At the conclusion of the experiment, rats were given a lethal dose of pentobarbital sodium and then thoracotomized and perfused with fixative for subsequent verification of vagotomy.

Lymph apoA-IV content was measured using an electroimmunoassay as previously described (13). Gels for electroimmunoassay were made of 1.2% indubiose agarose and 2% dextran in Tris-veronal buffer containing 1% goat anti-rat apoA-IV antiserum. Samples were subjected to electrophoresis at 3.5 V/cm for 6 h at 20°C. Immunoprecipitates were stained, and the areas under the precipitate rockets were measured using a computer-controlled digitizing system (SigmaScan, Jandel Scientific, Sausalito, CA).

Experiment 2: Effect of vagotomy on jejunomucosal synthesis of apoA-IV after duodenal infusion of triacylglycerol emulsion. Vagotomized and sham-vagotomized rats were given 8-h infusions of either glucose-saline or triolein emulsion into the duodenum. Previous studies (21) demonstrated that use of glucose-saline as a control infusate yielded the same results as sodium taurocholate in PBS and was less expensive. Jejunomucosal apoA-IV biosynthesis, based on the incorporation of [3H]leucine into immunoprecipitated apoA-IV as a percentage of incorporation of [3H]leucine into total mucosal protein (determined from incorporation into TCA-precipitable material), was measured as described previously (16).

Experiment 3: Effect of vagotomy on apoA-IV synthesis and mRNA levels in a jejunal Thiry-Vella fistula after ileal lipid infusion. Vagotomized and sham-vagotomized Thiry-Vella fistula rats were given 8-h infusions of either glucose-saline or fatty acid and monoglyceride emulsion into the ileum. Mucosal apoA-IV synthesis in the Thiry-Vella segment (~10 cm) was determined as described in Experiment 2. In addition, mucosa from a 1- to 2-cm segment of the same Thiry-Vella segment was processed for isolation of total RNA using the RNeasy RNA isolation kit (Qiagen, Valencia, CA), using the manufacturer's suggested protocol. Isolated RNA was stored at -80°C until analysis.

A full-length rat intestinal apoA-IV cDNA clone (pSP64AIV) was kindly provided by Dr. Jeffrey Gordon (Washington University, St. Louis, MO). A 438-bp fragment was obtained by digesting pSP64AIV with Sac I. The fragment was subcloned into pGEM-3Z (Promega, Madison, WI) to obtain pGEM3Z-AIV; this Sac I fragment was labeled with [alpha -32P]dCTP to a specific activity of 1 × 109 cpm/ng using the Ready-to-Go random primer oligo kit (Pharmacia, Piscataway, NJ). Northern blotting was performed as previously described (21). After autoradiography, blots were stripped and reprobed for 18S ribosomal RNA (Ambion, Austin, TX). Relative levels of apoA-IV mRNA in control (glucose-saline) vs. lipid-infused vagotomized or sham-vagotomized rats were determined by densitometric analysis, normalized to the 18S signal.

Experiment 4: Effect of vagotomy on apoA-IV synthesis and mRNA levels in a jejunal Thiry-Vella segment subjected to direct infusion of lipid. Vagotomized and sham-vagotomized Thiry-Vella fistula rats were given 8-h infusions of either glucose-saline or fatty acid plus monoglyceride emulsion directly into their Thiry-Vella segments, after which both mucosal synthesis and transcript levels of apoA-IV in the Thiry-Vella segment were measured.

Statistical Analysis

Data were analyzed using a commercial statistical software package (34). Time-dependent data on lymph flow and lymphatic output of lipids and apoA-IV in response to triolein in either vagotomized or sham-vagotomized rats (experiment 1) were analyzed as the change from baseline condition by two-way repeated-measures ANOVA. The effects of vagotomy on lipid-induced changes in apoA-IV synthesis and mRNA levels (experiments 2-4) were analyzed using two-way ANOVA. All post hoc testing was performed using a multiple general linear model. Differences were considered significant if P was <0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Experiment 1: Effect of Vagotomy on Lymphatic Lipid Transport and apoA-IV Secretion During Duodenal Infusion of Triacylglycerol

Body weights at the time of vagotomy were similar: 303 ± 11.5 g (sham) vs. 308 ± 8.1 g (vagotomized). Body weights were measured each day after initial surgery; by the sixth postoperative day, vagotomized rats lost an average of 21 ± 7.8 g, compared with a net gain of 10.4 ± 4.9 g in the sham rats.

Preliminary studies established that administration of fluorogold 3 days before lipid infusion studies had no effect on lymph flow, lipid transport, or apoA-IV output (data not shown).

Before lipid infusion, lymph flow was 1.9 ± 0.6 ml/h in the sham-vagotomized rats and 2.2 ± 0.4 ml/h in the vagotomized rats (not significantly different). In response to lipid infusion, lymph flow rate increased similarly in both groups; by the end of the infusion period (8 h), lymph flow was 3.6 ± 0.4 and 3.7 ± 0.7 ml/h in the sham and vagotomized groups, respectively.

With the onset of lipid infusion, radioactive lymph lipid output increased steadily in both groups, reaching a steady-state output of 60-70% of hourly infused radioactivity by 5 h. Over the course of the infusion period, there were no significant differences in lipid output between groups. After a characteristic lag over the first 4 h (16), lymph apoA-IV output also increased in response to lipid infusion from a basal level of ~100 µg/h to ~260 µg/h by 8 h. For the entire experimental period, lymphatic apoA-IV output remained slightly lower in the vagotomized group than in shams, but at no time point was this difference statistically significant.

Experiment 2: Effect of Vagotomy on Jejunomucosal Synthesis of apoA-IV After Duodenal Infusion of Triacylglycerol Emulsion

Neither vagotomy nor infusion had an effect on total protein synthesis in proximal jejunum (data not shown). There was no effect of vagotomy on synthesis of apoA-IV in proximal jejunal mucosa, either under control (duodenal infusion of glucose-saline) or lipid-infused (duodenal infusion of triolein emulsion) conditions (Fig. 1). In both groups, lipid infusion elicited a 2- to 2.5-fold increase in apoA-IV synthesis, compared with control infusion.


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Fig. 1.   Vagotomy has no effect on stimulation of apolipoprotein A-IV (apoA-IV) synthesis in proximal jejunum by duodenal infusion of triacylglycerol emulsion (experiment 2, see text). Vagotomized (vagx) or sham-vagotomized rats equipped with duodenal infusion cannulas received continuous, 8-h duodenal infusions of either glucose-saline (control) or lipid (triolein emulsion, 40 µmol/h). apoA-IV synthesis in proximal jejunal mucosa was then measured as described in MATERIALS AND METHODS. Values are means ± SE for 4 rats (sham, control), 6 rats (vagx, control), 5 rats (sham, lipid), and 7 rats (vagx, lipid). * Significant effect of lipid infusion on apoA-IV synthesis (P < 0.01).

Experiment 3: Effect of Vagotomy on apoA-IV Synthesis and mRNA Levels in a Jejunal Thiry-Vella Fistula After Ileal Lipid Infusion

Basal synthesis of apoA-IV in the jejunal Thiry-Vella loop was characteristically lower than in the corresponding segment of intact jejunum (Figs. 1 and 2); nevertheless, ileal lipid infusion elicited a twofold elevation in Thiry-Vella synthesis of apoA-IV in sham rats. This effect was blocked in the vagotomized group.


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Fig. 2.   Vagotomy prevents increase in jejunal apoA-IV synthesis in a jejunal Thiry-Vella fistula elicited by ileal lipid infusion (experiment 3, see text). Vagotomized or sham-vagotomized rats equipped with jejunal Thiry-Vella fistulas and ileal infusion cannulas received continuous, 8-h ileal infusions of either glucose-saline (control) or lipid emulsion (20 µmol/h monoolein, 40 µmol/h oleic acid). Mucosal apoA-IV synthesis in the Thiry-Vella segment was then measured as described in MATERIALS AND METHODS. Values are means ± SE for 6 rats (sham, control), 5 rats (sham, lipid), 6 rats (vagx, control), and 8 rats (vagx, lipid). * Significant effect of lipid (P < 0.001).

In the sham-vagotomized group, stimulation of apoA-IV synthesis in the Thiry-Vella segment by ileal lipid was not associated with a change in apoA-IV mRNA levels; similarly, vagotomy had no influence on apoA-IV mRNA in the Thiry-Vella mucosa (data not shown).

Experiment 4: Effect of Vagotomy on apoA-IV Synthesis and mRNA Levels in a Jejunal Thiry-Vella Segment Subjected to Direct Infusion of Lipid

Compared with control infusions, direct perfusion of the Thiry-Vella segment with lipid stimulated both apoA-IV synthesis (Fig. 3) and mRNA levels (Fig. 4); vagotomy did not influence this result.


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Fig. 3.   Vagotomy does not affect the stimulation of apoA-IV synthesis in a jejunal Thiry-Vella fistula in response to direct perfusion of lipid into the Thiry-Vella segment (experiment 4, see text). Rats prepared as described in MATERIALS AND METHODS (except no ileal cannula) received continuous, 8-h infusions of either control (glucose-saline) or lipid (20 µmol/h monoolein, 40 µmol/h oleic acid) directly into their Thiry-Vella segment. apoA-IV synthesis was then measured as described in MATERIALS AND METHODS. Values are means ± SE for 4 rats (sham, control), 3 rats (sham, lipid), 4 rats (vagx, control), and 4 rats (vagx, lipid). * Significant effect of lipid on apoA-IV synthesis (P < 0.05).



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Fig. 4.   Direct perfusion of a jejunal Thiry-Vella fistula with lipid stimulates expression of apoA-IV mRNA, independent of a vagal innervation (experiment 4, see text). Mucosal apoA-IV mRNA from Thiry-Vella fistulas of same rats as described in Fig. 3 was measured as described in MATERIALS AND METHODS. * Significant effect of lipid on apoA-IV mRNA levels (P < 0.01).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

There were three major findings in the present study. First, intestinal lipid transport and jejunal synthesis and secretion of apoA-IV in response to duodenal delivery of lipid were not dependent on intact vagal innervation. Second, stimulation of synthesis of apoA-IV in a jejunal Thiry-Vella fistula by ileal delivery of lipid was vagally mediated. Finally, whereas direct delivery of lipid increased both protein synthesis and mRNA levels of jejunal apoA-IV, the vagally mediated apoA-IV stimulatory ileal signal produced increases in apoA-IV synthesis that were unaccompanied by changes in steady-state mRNA levels. These results support the hypothesis that there are at least two separate mechanisms whereby intestinal lipid stimulates intestinal production of apoA-IV in the rat. In one, lipid in the proximal gut directly elicits an increase in apoA-IV gene expression, which is sufficient to account for the accompanying increase in apoA-IV protein synthesis. In the second mechanism, lipid in the distal gut elicits an indirect, vagally mediated signal, capable of increasing synthesis of apoA-IV at the posttranscriptional level.

Until recently (20), the only known stimulus for synthesis and secretion of intestinal apoA-IV was lipid transport (13, 16), in particular, the assembly and transport of chylomicrons by the enterocytes (13). Thus an effect of vagotomy on apoA-IV might be secondary to any effect of denervation on the lipid transport process. Steatorrhea is a common finding in vagotomized subjects (32), most likely due to the negative effect of vagotomy on the gastric grinding and sieving of solid food. However, previous studies in dogs have reported little effect of vagotomy on fat absorption (5, 8, 14) or on fat-stimulated hormone release (35), at least when liquid fat was used as the test meal (5). Similar information has not been available in the rat. Moreover, no previous study has specifically addressed the role vagal signals might play in the assembly and transport of chylomicrons. Our results support no such vagal role: intestinal lymphatic transport of duodenally infused lipid was unaffected by vagotomy, neither in the dynamic early phase occurring within the first 4 h after the start of lipid infusion nor in the later, steady-state phase of lipid transport. Vagotomy also had no effect on the coupling of jejunal apoA-IV synthesis and the apoA-IV output in lymph to lipid absorption. Thus the intestinal apoA-IV response to duodenally infused lipid is independent of vagal innervation.

Similar to the findings in duodenal infusion experiments (present study and Ref. 21), direct delivery of lipid emulsion into a jejunal Thiry-Vella fistula stimulated increases in mucosal synthesis and mRNA levels of apoA-IV in the Thiry-Vella segment, and these increases were not affected by vagotomy. Thus administration of lipid into the proximal intestine stimulates apoA-IV independently of vagal signals. The precise mechanism whereby lipid (presumably "directly") stimulates apoA-IV production and secretion remains unknown, although this coupling appears to depend specifically on the packaging and transport of chylomicrons (13).

In contrast to the effect of proximal gut lipid on apoA-IV, the ileal lipid-elicited signal that we (21) previously reported to stimulate jejunal apoA-IV synthesis does appear to depend on intact vagal innervation. Moreover, this signal has no influence on jejunal apoA-IV message levels, suggesting that it stimulates apoA-IV synthesis by a different mechanism than that apparently operating under conditions of direct proximal gut delivery of lipid.

Although these studies reveal a role for vagal signals in mediating the effect of ileal lipid on jejunal production of apoA-IV, the precise mechanism remains unclear. Previous findings obtained using capsaicin suggest that afferent signals are not involved in the effect, although the importance of such signals has not been completely ruled out (17). It also appears unlikely that CCK might be involved (17). At present, the most likely candidate in mediating the ileojejunal signal that stimulates apoA-IV synthesis appears to be the distal gut hormone, peptide tyrosine tyrosine [peptide YY (PYY)], which is currently thought to be a major endocrine mediator of the ileal brake (22). We (20) recently demonstrated that intravenously infused PYY, given at doses that reproduce plasma PYY levels observed after ileal lipid infusion, stimulates jejunal synthesis and lymphatic secretion of apoA-IV. Interestingly, PYY also had no effect on jejunal apoA-IV mRNA levels, making its effects similar to those of ileal lipid infusion. Although it remains to be determined whether PYY is involved in the effects of ileal lipid on proximal gut apoA-IV production, there is now substantial indirect evidence supporting such a role. First, in rats, apoA-IV has been shown to have PYY-like effects, particularly in inhibition of gastric acid secretion (23) and inhibition of gastric emptying (24). Second, similar to the effects of distal gut lipid (22) and PYY (2, 15, 25, 26, 27, 29) on gastric acid and pancreatic exocrine secretion, we have now demonstrated that stimulation of jejunal production of apoA-IV by ileal lipid infusion is blocked by vagotomy. Third, similar to the aforementioned effect of PYY, this vagally mediated signal stimulates synthesis of jejunal apoA-IV without an accompanying increase in apoA-IV mRNA levels. Finally, recent preliminary studies have shown that central administration of neuropeptide Y, which has strong affinity for the same receptors as PYY, stimulates jejunal synthesis of apoA-IV (4). It remains to be determined whether the effects of PYY on apoA-IV are vagally dependent or, indeed, whether endogenous PYY mediates the effect of ileal lipid on jejunal apoA-IV synthesis. Further work is necessary to examine these questions.

In previous studies (31), we demonstrated that, after rats received a gastric bolus of fat, there is a rapid (i.e., within 15-30 min) increase in intestinal mucosal synthesis and lymphatic output of apoA-IV. This was correlated with a similarly rapid spread of administered lipid throughout the gut, including the ileum and cecum. Significantly, the mass of lipid present in the distal gut was similar to the amounts present after ileal lipid infusion under conditions identical to those used here (21). We (19, 31) proposed that, on ingestion of a high-fat meal, rapid spread of lipid throughout the gut, likely due to initial "dumping" from the stomach in the earliest phases of a meal before the activation of regulatory mechanisms controlling gastric emptying, could result in a rapid apoA-IV stimulatory signal. In view of the fact that lymphatic output and plasma levels of apoA-IV rise significantly within 30 min after a fat meal (31), some sort of rapid response mechanism is implied. Such a mechanism would be consistent not only with the rapid rise in intestinally secreted apoA-IV, sufficient enough to produce plasma levels of this protein capable of inhibiting food intake (9, 31), but also with the recently proposed role of apoA-IV as an endogenous, lipid-induced, postprandial antioxidant (30).

The pattern of the jejunal apoA-IV response to ileal lipid in this study (i.e., stimulation of synthesis without a change in apoA-IV mRNA levels) is similar to that seen in the immediate response to a gastric lipid bolus (18, 31) but different from that observed in response to slow, continuous duodenal lipid infusion, which is characterized by similar magnitude increases in both apoA-IV synthesis and mRNA levels (16). Moreover, ileal lipid infusion produces levels of ileal and cecal luminal and wall lipid (21) similar to those observed in response to gastrically delivered lipid meals (31), whereas duodenal infusion results in virtually no lipid reaching the distal gut (16). These observations suggest that the mechanism whereby ileal lipid infusion stimulates jejunal apoA-IV production may be similar to that involved in the early apoA-IV response to a gastric lipid bolus, and thus the ileally infused, Thiry-Vella fistula model may induce mechanisms that simulate events occurring early after a lipid meal.

Previously available evidence has supported the hypothesis that apoA-IV is controlled mainly at the pretranslational level (1, 11, 21). A novel implication of the present work is that it suggests the existence of at least two fat-inducible mechanisms: one depending on increases in mRNA levels and a separate, posttranscriptional mechanism capable of upregulating apoA-IV production without a change in gene expression. Indeed, we have recently shown that a gastric fat bolus rapidly produces significant increases in intestinal apoA-IV synthesis within 30 min (31), several hours before mucosal apoA-IV mRNA levels begin to show an increase. This rapid rise in apoA-IV synthesis was not blocked by the transcriptional inhibitor actinomycin D. Moreover, within 30 min, there was a clear shift in the distribution of mucosal apoA-IV mRNA from the unbound ribonucleoprotein to the polyribosome fraction of cytoplasm (18). These results suggest a rapid, lipid-induced, translational mechanism for control of apoA-IV at the level of initiation. Details of this mechanism await further elucidation. Moreover, further work is necessary to determine if a similar mechanism is involved under conditions used in the present study and in response to PYY.


    ACKNOWLEDGEMENTS

We thank Dr. Maria-Dolores Rodriguez for helpful suggestions during the initial phases of this investigation; we are also grateful to Elizabeth Baronowsky and Dr. Terry Powley for teaching us the procedures for vagotomy and vagotomy verification.


    FOOTNOTES

These studies were supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants to T. J. Kalogeris (DK-08828 and DK-52148) and to P. Tso (DK-32288 and DK-53444) and by funds from the Louisiana State University Medical Center Department of Surgery.

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: T. J. Kalogeris, Dept. of Surgery, LSU Medical Center, 1501 Kings Highway, Shreveport, LA 71130 (E-mail: tkalog{at}lsumc.edu).

Received June 14 1999; accepted in final form 10 August 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Gastroint Liver Physiol 277(5):G1081-G1087
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