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APPETITE, OBESITY, DIGESTION, AND METABOLISM

NT-4-deficient mice lack sensitivity to meal-associated preabsorptive feedback from lipids

Department of Psychological Sciences, Integrative Program in Neuroscience, and Ingestive Behavior Research Center, Purdue University, West Lafayette, Indiana

Submitted 22 November 2006 ; accepted in final form 8 February 2007

	ABSTRACT

TOP ABSTRACT EXPERIMENT 1. DEVELOPMENT AND... EXPERIMENT 2. SATIATING EFFICACY... EXPERIMENT 3. SATIATING EFFICACY... GENERAL DISCUSSION APPENDIX A APPENDIX B GRANT REFERENCES

 
Since mice with a deletion of the neurotrophin-4 (NT-4) gene exhibit a loss of both nodose ganglion neurons and vagal afferent terminals in the small intestines, we hypothesized that the reduced intestinal innervation of the NT-4 knockout (NT-4KO) mouse would lead to a corresponding reduction in the preabsorptive feedback from macronutrients. To explore this prediction, we measured meal patterns in NT-4KOs and controls, while, on different days, intragastric infusions of either lipids (Intralipid; 10%, 20%) or glucose (12.5%, 25%) were yoked to each animal's spontaneous feeding of a pelleted diet (1 kcal infused/1 kcal ingested). NT-4KO mice were relatively, though not completely, insensitive to the lipid infusions, whereas they were as sensitive as controls to glucose infusions. More specifically, the regulatory deficits of NT-4KOs included 1) attenuated satiation from the lipid infusions, as measured by smaller intrameal reductions of both meal size and meal duration, 2) defects in satiety associated with the fat infusions, as measured by smaller intermeal increases of both satiety ratio and intermeal interval, and (3) losses in daily compensatory responses for lipid calories. These results support the hypothesis that NT-4KO mice have deficits in macronutrient feedback from the gastrointestinal tract, indicate that the defects are specific insofar as they do not include impairments in the feedback of glucose infusions on feeding, and suggest that early feedback about dietary lipids is important in the regulation of satiation, satiety, and longer-term compensation of daily caloric intake.

satiation; meal analysis; vagus nerve; Intralipid; glucose

Feeding, or energy intake, is controlled in part by early, preabsorptive feedback produced by nutrients within the gastrointestinal (GI) tract. Studies have indicated that this negative feedback from macronutrients can differentially affect satiation (or the processes that terminate a meal; usually a result of preabsorptive, immediate effects of the nutrients) and satiety (or the processes that delay meal onset; often assumed to be the later, postabsorptive effects of a meal) and engage different metabolic and regulatory mechanisms. The main purpose of the present series of experiments was to investigate, using the NT-4KO mouse, the contribution of NT-4-dependent vagal afferents to within (or intrameal) feedback or satiation effects and between (or intermeal) satiety effects. Such effects were examined for two representative macronutrients, the lipid mixture Intralipid as a representative fat and glucose as a carbohydrate.

	EXPERIMENT 1. DEVELOPMENT AND STANDARDIZATION OF A PARADIGM YOKING GASTRIC NUTRIENT INFUSIONS TO AD LIBITUM FEEDING

TOP ABSTRACT EXPERIMENT 1. DEVELOPMENT AND... EXPERIMENT 2. SATIATING EFFICACY... EXPERIMENT 3. SATIATING EFFICACY... GENERAL DISCUSSION APPENDIX A APPENDIX B GRANT REFERENCES

 
Experiments that have evaluated the effects of nutrient infusions on ingestive behavior have commonly employed fixed-volume and arbitrarily timed preloads (e.g., 5, 13). To reduce the arbitrariness of both the volume infused and the timing, as well as to maximize the temporal contiguity between meal taking and infusions for the present series of experiments, we adapted an intake-monitoring-and-infusion paradigm. In this protocol, discrete, small-volume intragastric (IG) infusions of lipids (or other experimental solutions) were yoked to the animals' spontaneous ad libitum consumption of a balanced pelleted maintenance diet. The design also made it practical to simultaneously perform meal-pattern analyses to assess the impacts of different infusions.

Given the limited availability of NT-4KO mice, this initial series of standardization trials and analyses was performed with the more readily available and extensively used C57BL/6 strain of mouse. This made it practical first, to determine effective infusion parameters and second, to verify the feasibility and utility of the yoked infusion paradigm. These standardization experiments with C57BL/6 mice also made it more practical to evaluate whether the volume and rates of infusate delivery produced any nonspecific and nonnutritive effects on meal taking; for this assessment, the effects of saline vehicle infusions were compared with sham infusions. Finally, as well as establishing baselines, an additional advantage of using the C57BL/6 strain was that it provided another genetic background against which the "wild type" genotype of the S129 mouse, the control background strain for the NT-4KO mice used in experiments 2 and 3, could be evaluated.

Methods

Animals. Male C57BL/6 mice, 7–8 wk old (n = 8 for saline vs. sham no-infusion comparison; n = 15 for Intralipid infusions; Harlan Industries, Indianapolis, IN), weighing 20–25 g upon arrival, were housed individually, maintained on a 12:12-h light-dark schedule (lights on at 2400) at 23°C, and initially given ad libitum access to tap water and chow [60% carbohydrate, 28% protein, 12% fat (Laboratory Rodent Diet 5001; PMI Feeds, Brentwood, MO)]. Body weights were recorded daily. All protocols were conducted in accordance with the Principles of Laboratory Animal Care (National Institutes of Health publication no. 86–23, revised 1985) and the guidelines of the American Association for Accreditation of Laboratory Animal Care and were approved by the Purdue University Animal Care and Use Committee.

Acclimation/diets. In preparation for the infusion trials, mice were first adapted to the test diet and housing conditions. On being introduced to their cages, the mice were maintained on the PMI maintenance diet available in a food hopper for 2–3 days. They were then given access to both the maintenance chow diet and the test diet (20 mg pellets; 66% carbohydrate, 22% protein, 12% fat; product no. F0071; Bio-Serv, Frenchtown, NJ) until the majority of their intake consisted of pellets, after which the mice were maintained on just the pellets. After 2–3 days of exclusively consuming pellets from an open feeding jar, mice were fed with automated pellet dispensers (Coulbourn Instruments, Allentown, PA). Details concerning the apparatus were previously described (3).

Surgery. Once pellet intakes had stabilized, each mouse was laparotomized and surgically implanted with an indwelling Silastic catheter, internal diameter = 0.025" (Dow Corning, Midland, MI) in the greater curvature of the stomach. The catheter was anchored to the stomach with a stay suture and Marlex mesh, and then closed with a pursestring suture at the entry point of the stomach, as well as with a serosal tunnel around the catheter at the fundus of the stomach. The catheter was then passed through the abdominal wall and passed subcutaneously to a back mount (Instech Solomon, Plymouth Meeting, PA) and capped. Once the animals had recovered to at least presurgical body weights, each animal's catheter was connected by an infusion line and overhead swivel to an infusion pump. Animals then remained attached to their infusion lines until the end of the experiment, eliminating handling disturbances of the mice after the initial hookup and also allowing for monitoring of feeding and administrating of infusions in the home cage.

Testing. Animals were able to free-feed pellets for 20 h per day in their home cages, beginning at the onset of the dark phase (1200) and ending after 8 h of the light phase (0800). During a 4-h period of no feeding (between 0800 and 1200), the equipment was maintained and the previous day's data were exported for analysis. Animals were tested for 18 days in the first part of the experiment (alternating 3-day blocks of saline infusions and sham no infusions) or for 30 days in the second part of the experiment (Intralipid series) so that multiple infusions of each concentration of fat could be administered.

Infusates/infusion pumps. Infusion conditions were varied daily such that a single infusate was yoked to pellet intake over the entire 20 h of a test day. Infusions consisted of 1) vehicle/volume controls of 0.9% saline, 2) sham no-vehicle infusions in which no infusate was delivered, 3) 10% Intralipid (Baxter Healthcare, Deerfield, IL) (1 kcal/ml), and 4) 20% Intralipid (2 kcal/ml). The four infusion conditions were administered according to the following schedules: In the first portion of the experiment, each pellet consumed was yoked to sham no-vehicle infusions for three consecutive days, followed by 3 days of yoked saline infusions. This was repeated three times until each condition (saline or no-vehicle infusion) was completed, for a total of 9 days.

For the second portion of the experiment, the two concentrations of fat were each administered three times, with at least 2–3 days of saline infusions interspersed between each fat-infusion day. The order of fat infusions was semirandom, that is one concentration was randomly chosen to be infused on one test day while the other concentration would be infused on a later test day after the corresponding saline infusions. This was repeated for each of the three sets of infusions, so that each concentration of fat was administered three times.

Selectable-speed infusion pumps (Razel; integrated with the Coulbourn feeding equipment) were used to deliver infusates. A computer monitored the status of an infrared beam (interrupted by a single pellet in the trough) every 20 ms and, upon removal of a pellet, another pellet was dispensed. As each pellet was removed and consumed by the animal, 37 µl of infusate was delivered intragastrically over 2 s. Each 20-mg pellet contained 0.068 kcal, and each 2-s infusion yielded either 0.037 kcal of 10% Intralipid or 0.074 kcal of 20% Intralipid. Consumption was verified by checking daily for pellets at the bottom of each cage. The presence of pellets indicated either incomplete consumption or hoarding of pellets, and data for that animal were subsequently not used.

Data Analysis

Intrameal and intermeal effects of infusions on ad libitum feeding. The start of a meal was defined as the beginning of a period in which three pellets were consumed within a 7-min interval, while the end of a meal was defined as the beginning of a 10-min period of no eating after a meal had been initiated; these criteria were based on previous experiments and validations of meal patterning criteria with similar feeding systems (Ref. 3; see also Appendix A).

Average meal parameter changes were calculated for both the 12-h dark phase and the entire 20-h daily test period for a given infusion condition. In addition, the first three self-paced meals of each daily trial were also compared. Since the first meal represented the first infusion of a given 20-h trial (also the first meal of the dark phase and the first meal after the 4-h deprivation), this bout of feeding should have reflected primarily any early, presumably intrameal preabsorptive effects of the infusate on feeding. In contrast, the second and third meals that followed might have reflected longer-term intermeal cumulative effects of the first meal infusion, as well as the infusions that accompanied the second and third meals. Expressed in terms of possible outcomes, if the intragastric infusions of macronutrients affected primarily preabsorptive (or "direct") (28) feedback and short-term satiation, conceivably no cumulative effects of the infusions across meals would occur and each of the first three meals of the day would evidence similar changes in the different meal parameters. In contrast, if the infused macronutrients had postabsorptive and longer-term (or "indirect") (28) effects that influenced satiety as well, the different meal parameters of the third (or second and third) meals would be expected to reflect cumulative effects of the previous infusions.

Statistics. All comparisons for the Intralipid vs. saline infusions were made by first averaging the values for each animal, then averaging those values to obtain averages for each infusate and group. Results were analyzed using one-way ANOVAs and any significant values were further examined with Tukey's post hoc tests. Comparisons for saline vs. sham no-infusion conditions were analyzed using independent t-tests. Significance was considered as a P value of <0.05.

Results

Intrameal effects of infusions on ad libitum feeding. Intralipid infusions were effective in reducing the size of meals as well as influencing other within-meal measures of intake in the C57BL/6 mice. For example, meal size was significantly decreased by both of the concentrations of lipid infusions, and this decrease was observed for each of the first three individual meals (P < 0.001 for each of the three meals), as well as for the overall dark phase (P < 0.05) and 20-h daily (P < 0.05) means. There was also a consistent dose response effect in which 20% Intralipid reliably produced a greater reduction in meal size (e.g., 30% in the dark phase; see Table 1) than 10% Intralipid (e.g., 18% in the dark phase) (P < 0.05). Both the reduction in meal size and the dose response pattern observed were generally paralleled by similar decreases in meal duration (see Table 1). Overall, t-tests revealed that most intrameal parameters differed between the two concentrations of Intralipid, and a similar pattern of results was obtained for the individual meals of the early dark period, as well as for the means of the 12-h dark phase and the 20-h daily means.

Daily caloric compensation for infused Intralipid. With Intralipid infusions, the mice did not reduce their pellet consumption sufficiently to maintain strict stability of daily caloric intake. However, total caloric intakes (pellets plus infusate) were not significantly different between any of the infusate conditions during the dark phase or 20-h daily period, indicating that any undercompensation to the lipid infusions was minor (Fig. 1).

View larger version (17K): [in this window] [in a new window]   Fig. 1. Total caloric intakes (means ± SE) during the 20-h daily period (A) and the 12-h dark phase (B) for C57BL/6 mice (n = 15) after Intralipid infusions. Total calories consisted of calories from the Intralipid infused and the pellets consumed. The caloric intakes did not vary between any of the infusates (saline vs. 10% Intralipid vs. 20% Intralipid), indicating that any undercompensation of fat calories infused was minor.

Body weight changes over the test period. Body weights increased an average of 25% from the beginning of the experiment (day 0, when the mice were connected to the apparatus postsurgery) to the end of the experiment. Importantly, based on the final weights after 30 days of testing, no animal lost weight over the course of the experiment. Based on growth curves for C57BL/6 mice of this age range, the growth was typical (20%, Harlan); it was also in line with our own observations of body weight increases in the lab for this strain (Chi MM and Powley TL, unpublished observation).

Discussion

The present experiment established a practical protocol for evaluating the sensitivity of mice to GI infusions of lipids and yielded a basic dose response series for the yoked infusion paradigm. In an infusion study that approximated the conditions of the present experiment in key respects, Greenberg et al. (9) observed that Intralipid infusions into the duodenum in rats first lead to increases in absorbed fats in the circulation 30 min after the infusion. Although, to the best of our knowledge, no precise determinations are available for the rate at which the lipid constituents of Intralipid are absorbed from the GI tract of mice, the 30-min estimate from the Greenberg experiment seems a reasonable approximation for mice in the yoked infusion paradigm.

Thus, the 30-min estimate for the onset of blood lipid elevation provided by the experiment of Greenberg and co-workers (9) may be a useful estimate of the period during which the effects of fats would be predominately preabsorptive (i.e., the 30 min from the beginning of infusions) and the time point (> 30 min) at which more postabsorptive effects might begin to accumulate. Using this estimate in conjunction with the distinction that "satiation" refers to the process(es) that terminate a bout of feeding and that "satiety" denotes the process(es) that inhibit the initiation of a following meal, it is possible to formulate tentative conclusions concerning the pathways and mechanisms whereby the Intralipid infusions in the present experiment produced nutrient-related negative feedback limiting food intake.

Increased satiation was reflected in the decreases in intrameal parameters, including meal size and meal duration, associated with the lipid infusions. Such effects occurred sufficiently rapidly (e.g., meal durations in the dark phase averaged 3.98 min with 20% Intralipid and 4.65 min with 10% Intralipid, compared with 5.13 min after saline infusions), that the effects would, in all likelihood, be preabsorptive and mediated by vagal afferents (14, 17). Increased satiety, on the other hand, was reflected in the increased satiety ratios and IMIs produced by the lipid infusions. The proportionately greater delays in the initiation of the next meal following a meal of a particular size were sufficiently protracted that postabsorptive feedback mechanisms may have been engaged (e.g., 20% Intralipid infusions for the dark phase increased the satiety ratio by 164%, and the IMI was lengthened to 45 min compared with 24 min after saline infusions). Thus, the observed intermeal effects are likely to have occurred when some postabsorptive effects of the lipid infusions were operating in conjunction with the preabsorptive feedback detected with the intrameal feeding parameters.

The fact that the impact of the lipid infusions was limited to the intrameal and early intermeal processes and did not appear to accumulate over successive meals (cf. the analyses of the first three meals of the dark phase in Table 1) suggests that the pre- and postabsorptive feedback effects of the Intralipid infusions delivered according to the present yoked infusion protocol tend to dissipate or to have half-lives that can be measured in tenths of a minute rather than hours.

The fact that vehicle (saline) infusions did not differ from no-infusate sham infusions indicates that the volume and rate of the infusions did not trigger nonnutritive nonspecific suppression of feeding. The apparent but nonsignificant trend of the saline infusions to reduce some of the meal-taking parameters suggests, though, that the volume and rate combination employed may have been approaching a threshold and that higher volumes and/or rates might well begin to produce distension or nonnutritive effects on feeding behavior (see, for example, 1, 8, 11).

	EXPERIMENT 2. SATIATING EFFICACY OF FAT INFUSIONS IN S129, NT-4KO MICE

TOP ABSTRACT EXPERIMENT 1. DEVELOPMENT AND... EXPERIMENT 2. SATIATING EFFICACY... EXPERIMENT 3. SATIATING EFFICACY... GENERAL DISCUSSION APPENDIX A APPENDIX B GRANT REFERENCES

 
Experiment 1 established the efficacy and practicality of the protocol yoking infusions of nutrients directly into the stomach with ad libitum, self-paced consumption of small (20 mg) test pellets. The present experiment employed the same yoked infusion protocol to evaluate the effect of the Intralipid infusions on both short-term, or meal-by-meal, and long-term, or 12-h dark-phase and 20-h daily, feeding patterns in NT-4KO mice.

Since converging electrophysiological (14, 18, 24) and physiological (15, 17, 27) evidence indicates that vagal afferents to the intestines detect lipids and since, as already reviewed, the deletion of the NT-4 gene product in the NT-4KO mouse produces a loss of vagal innervation of the small intestine smooth muscle, as well as a loss of the fibers that course through the muscle layers to innervate the mucosa (cf. 7), we hypothesized that NT-4KO mice might prove less sensitive to lipid infusions than their S129 controls. To examine this hypothesis we evaluated the sensitivity of NT-4KO and S129 (the genetic background for the knockout) control mice to lipid infusions into the GI tract, using both the same concentrations of Intralipid and the same intake-yoked infusion protocol standardized in the previous experiment.

Methods

Animals. Animals consisted of male NT-4-deficient mice (NT-4KO), 12–16 wk old (129S4/SvJae-Ntf5^tm1Jae; –/–) (n = 12; Jackson Laboratory, Bar Harbor, ME) and wild-type mice, 12–16 wk old (129S3/SvImJ; +/+) (n = 12, Jackson Laboratory) weighing 20–30 g at the start of the experiment. [Availability issues made it impractical to match the ages of NT-4KOs and S129 controls with the ages of the C57BL/6s in experiment 1.] All animals were housed and maintained in a manner identical to those in experiment 1. Acclimation, diets, surgery, and testing for all mice were identical to experiment 1 except the sham no-infusion was not investigated, animals were tested for 30 days so that multiple infusions of each concentration of fat could be used, and saline was used as the sole control for the fat infusates.

Data analysis. Similar analyses were performed as in experiment 1, except values for comparing between strains were analyzed using two-way ANOVAs, with "group" and "concentration" as the independent variables and any significant values were further examined with Tukey's post hoc tests.

Results

Intrameal effects of infusions on ad libitum feeding. In general, both S129 controls and NT-4KO mice showed intrameal effects of the lipid infusions for most of the parameters examined. S129 mice, for example, had reduced meal sizes after Intralipid infusions that were significant for meal 2 (P < 0.001) and meal 3 (P < 0.005) as well as the dark phase (P < 0.005) and the 20-h daily (P < 0.005) means. The two concentrations of infusate differentially affected the parameters, as evidenced by a larger effect of the 20% Intralipid on meal size (e.g., 22% in the dark phase; see Table 2) vs. 10% Intralipid (e.g., 13% in the dark phase) (P < 0.05). Similar trends were seen in reductions of meal durations for the individual meals as well as the dark phase and 20-h daily means.

Intermeal effects of infusions on ad libitum feeding. As with the C57BL/6 mice (in experiment 1), S129 mice also showed effects of the lipid infusions on most of the intermeal measures. For example, satiety ratio was increased for meal 2 (P < 0.01) and meal 3 (P < 0.001), as well as for the overall dark phase (P < 0.001) and 20-h daily (P < 0.001) means, and similar trends were seen for increases in IMI. In addition, both parameters were significantly larger after 20% infusions compared with the 10% infusions (P < 0.001).

The comparison of most interest revealed that NT-4KO mice showed much smaller effects of the infusions, as they had attenuated increases in dark-phase IMI (P = 0.055) and satiety ratio (P < 0.001) compared with the S129 mice. Meal parameters for the first three meals, the average dark phase, and total daily parameters are summarized in Table 2.

On most measures, S129 mice showed percent changes similar to those of the C57BL/6 mice of experiment 1. Most differences between the two strains were due to an increased sensitivity of the C57BL/6 mice (e.g., larger satiety ratio) after 20% Intralipid infusions; otherwise the percent changes from saline baseline were comparable.

Concentration effects for some meal parameters (e.g., meal duration) in S129 and NT-4KO mice were not as evident with the first meal of the different Intralipid trials as they were with the C57BL/6 mice. However, by meals two and three the effects were more apparent. While this could reflect a strain difference, the most likely explanation is the larger overall intakes of the C57BL/6 mice, which likely reflects their younger age and continued growth. With a higher saline baseline, changes due to fat infusions would be more easily detected and would be reflected as a larger percent change, as was evidenced in most of the parameters of the C57BL/6 mice. Regardless, when accounting for all the meals in a day, percent changes for average dark phase and 20-h daily means were similar in S129 and C57BL/6 mice.

Daily caloric compensation for infused Intralipid. As would be expected from the results of the different meal parameters, total caloric intakes (kcals from pellets plus kcals from infusate) on lipid infusion days were significantly higher for NT-4KO mice than for the S129 controls. While S129 mice undercompensated slightly for the lipid infusions and tended to go into a positive energy balance, they had only nonsignificant increases in intake after 10% and 20% Intralipid infusions for both the 20-h daily (P > 0.05) and dark phase (P > 0.05) periods. In contrast, and consistent with a loss of longer term feedback, however, NT-4KO mice had larger (35%) overconsumptions of total calories in the 12-h dark phase and the 20-h daily periods (P < 0.001) (Fig. 2).

View larger version (20K): [in this window] [in a new window]   Fig. 2. Total caloric intakes (means ± SE) during the 20-h daily period (A and C) and the 12-h dark phase (B and D) for S129 mice (n = 12) and neurotropin-4 knockout (NT-4KO) mice (n = 12) after Intralipid infusions. The S129 mice (A and B) did not vary in their caloric intakes, regardless of the infusate (saline, 10% Intralipid, 20% Intralipid), indicating they compensated for the calories from the Intralipid infusions by reducing pellet intake. NT-4KO mice (C and D), however, undercompensated for the Intralipid infusions which led to a large overconsumption of total calories after fat infusions. *Significant difference (P < 0.05) from saline infusions.

Discussion

The present experiment supported the findings of a dose response effect of the Intralipid infusions by using the S129 mouse, which served not only as another control strain, but specifically as the background strain for the NT-4KO model. It also tested the hypothesis that vagal afferents play a significant role in providing feedback of lipids in the GI tract. NT-4KO mice, with a selective loss of vagal afferents to the intestines, showed significantly attenuated sensitivity to fat infusions in key intrameal, intermeal, and 20-h daily measures.

Analysis of meal patterning in the S129 background strain confirmed that the lipid infusions had immediate intrameal effects that could be observed with decreases in meal size and duration (e.g., 20% Intralipid resulted in average meal sizes of 5.5 pellets in the dark phase compared with 7.1 pellets after saline infusions). In addition, intermeal effects were also observed with increases in satiety ratio and IMI (e.g., 20% Intralipid resulted in an average IMI of 44 min in the dark phase compared with 30 min after saline infusions).

Interestingly, NT-4KO mice were relatively, though not completely, insensitive to the intake-yoked Intralipid infusions. The attenuated sensing of the NT-4KOs appeared in three different indexes. First, the NT-4KOs showed little or no suppression of the intrameal measures of meal size and meal duration during the first three meals of the infusion trial days. This pattern was also similar over the infusion trials: for instance, the average dark-phase meal size was 3.3 min after saline infusions, 3.1 min after 10% Intralipid, and 3.4 min after 20% Intralipid infusions. Second, compared with those of the S129 controls, the intermeal satiety ratio indices of NT-4KOs also evidenced an attenuated sensitivity to lipid infusions. Third, whereas the S129 controls (and the C57BL/6 animals in experiment 1, as well) decreased their 20-h pellet intakes on lipid infusion trials sufficiently to remain in the same net energy balance that they achieved on saline infusion days, the NT-4KOs failed to reduce their pellet intakes sufficiently over 20-h trials with yoked lipid infusions, thus going into a net positive energy balance (see Fig. 2).

The NT-4KOs did show some residual responses to lipid infusions. In the analyses of meal-to-meal cumulative effects of the infusions, sometime after the third meal, they began to show small suppressions of intermeal effects. For example, the average dark-phase satiety ratio increased from 4.6 min for saline to 5.2 min and 7.5 min for 10% and 20% Intralipid, respectively. This indicates that, although NT-4KO mice could not detect the immediate arrival of lipids, they were able to detect some of the cumulative or later effects. It has already been established that NT-4KO mice have a large reduction (50%) of nodose ganglion neurons and a larger loss (80%) of vagal fibers traversing the muscle layer of the GI tract, presumably en route to the mucosal layer where nutrients are detected. The fact that these losses are not complete, however, is consistent with the finding that the NT-4KO mice would continue to exhibit decreases in the intrameal parameters of meal size and duration and that intermeal processes continued to produce increases in satiety ratio and IMI, but with the smaller magnitudes observed.

	EXPERIMENT 3. SATIATING EFFICACY OF GLUCOSE INFUSIONS IN C57BL/6, S129, AND NT-4KO MICE

TOP ABSTRACT EXPERIMENT 1. DEVELOPMENT AND... EXPERIMENT 2. SATIATING EFFICACY... EXPERIMENT 3. SATIATING EFFICACY... GENERAL DISCUSSION APPENDIX A APPENDIX B GRANT REFERENCES

 
Experiment 2 established that NT-4KO mice display a reduced sensitivity to meal-yoked lipid infusions, both over the course of individual meals and over 20-h periods of testing. In view of this deficit or deficits in nutrient sensing, it seemed relevant to evaluate the specificity of the losses produced by deletions of NT-4 by investigating the effect of infusions using a second class of macronutrients.

Simple carbohydrates, in particular a sugar, such as glucose, should provide useful stimuli to probe the sensitivity of the NT-4KO mouse, since numerous arguments support two competing but plausible predictions. Given the ready uptake and absorption of glucose by the small intestine, a credible argument is that handling and utilization of the sugar is so rapid (1, 21) that most, if not all of its negative feedback on energy handling would be mediated by postabsorptive mechanisms as opposed to vagal afferents in the intestines. Conversely, a number of experiments, including single-unit electrophysiological studies, have concluded that a subset of vagal afferents are sensitive to glucose administered into the GI tract (6, 20), thus suggesting that a simple sugar would provide nutrient feedback that could control meal-taking or short-term feeding behavior, as observed with lipid infusions.

To evaluate these issues, the present experiment employed the identical yoked infusion, ad libitum feeding paradigm used in experiments 1 and 2, but used glucose rather than lipids as the infusate. As in the earlier experiments, the doses of glucose were first standardized using readily available C57BL/6 mice, and these doses were then used with S129 and NT-4KO mice. This sequence also allowed for a comparison of the C57BL/6 and S129 mice.

Methods

Animals. All animals were housed and maintained in a manner identical to experiments 1 and 2. Animals consisted of naïve male C57BL/6 mice, 7–8 wk old (n = 14), weighing 20–25 g upon arrival; naïve male NT-4-deficient mice (NT-4KO), 18–19 wk old (n = 7); and naïve wild-type S129 mice, 18–19 wk old (n = 7) weighing 20–30 g at the start of the experiment. Acclimation, diets, surgery, testing, and data analysis for all mice were identical to experiment 2, except 12.5% and 25% glucose (Mallinckrodt Laboratory Chemicals, Phillipsburg, NJ) were substituted as the infusates on nonsaline days. Each 2-s infusion yielded 0.018 kcal of 12.5% glucose or 0.037 kcal of 25% glucose delivered in volumes of 37 µl per 2-s infusion. (The viscosity of more concentrated glucose solutions limited the range of practical concentrations.)

Results

Intrameal effects of glucose infusions on ad libitum feeding. As they did to Intralipid infusions (cf. experiment 1), C57BL/6 mice showed reductions in intrameal effects after the glucose infusions but of a smaller magnitude. While this could have been due to a number of factors, one possibility is that the caloric densities of the glucose infusates were smaller (0.5 and 1.0 kcal/ml) compared with Intralipid (1.0 and 2.0 kcal/ml). Meal size was decreased during meal 1 (P < 0.001), as well as overall dark phase (P < 0.01) and 20-h daily (P < 0.001) periods. As an example of the dose-dependent suppression that resulted, 25% glucose reduced meal size by a larger extent (e.g., 22% in the dark phase, see Table 3) compared with 12.5% glucose (e.g., 18% in the dark phase) (P < 0.01). Similar reductions were observed for meal duration. The t-tests revealed that most of the intrameal parameters were different between the two concentrations of glucose, and similar trends were observed for the individual meals of the early dark period, the 12-h dark phase, and the 20-h daily means (Table 3).

Intermeal effects of glucose infusions on ad libitum feeding. C57BL/6 mice also showed increases in intermeal effects of the glucose infusions, as seen with increases in satiety ratio for meal 1 (P < 0.05), as well as the dark phase (P < 0.001) and 20-h daily (P < 0.001) periods. Similar trends were observed for IMI in the dark phase and 20-h daily period, as 25% glucose resulted in larger increases than 12.5% glucose (P < 0.01). The meal parameters for the first three meals, the average dark phase, and 20-h daily means are summarized in Table 3.

S129 mice also showed increases in intermeal effects of the glucose infusions, as seen with increases in satiety ratio for the dark phase (P < 0.001) and the 20-h daily (P < 0.01) periods. They also showed dose-dependent effects of the glucose infusions, with 25% glucose resulting in larger increases in IMI and satiety ratio (P < 0.05).

NT-4KO mice, as they also evidenced in their intrameal responses, did not show attenuated changes in satiety ratio or IMI compared with the S129 mice, as their changes were similar in magnitude to those of the S129 mice. In fact, only the 20-h satiety ratio means were significantly different (P = 0.025) and the NT-4KO mice had larger satiety ratios, which is in the opposite direction from the pattern of attenuation observed with the Intralipid infusions. The meal parameters for the first three meals, the average dark phase, and 20-h daily means are summarized in Table 4.

Overall daily caloric compensation for infused glucose. As was also observed with Intralipid infusions (cf. experiments 1 and 2), C57BL/6 mice did not reduce their pellet intakes precisely enough to compensate for the calories infused. However, unlike the Intralipid infusions, baseline intakes of glucose infusions were slightly but significantly larger than saline infusions during the dark phase (Fig. 3) (P < 0.05). The S129 mice showed somewhat larger caloric intakes, as 12.5% glucose resulted in a larger caloric intake for the 20-h daily period (P < 0.05) but not for the dark phase, and 25% glucose resulted in larger caloric intakes for both time periods. NT-4KO mice, unlike with lipid infusions, did not have any significant differences in caloric intake for any of the infusates or time periods (Fig. 4) (P > 0.05).

View larger version (16K): [in this window] [in a new window]   Fig. 3. Total caloric intakes (means ± SE) during the 20-h daily period (A) and the 12-h dark phase (B) for C57BL/6 mice (n = 14) after glucose infusions. C57BL/6 mice did not vary in their caloric intakes regardless of infusate during the 20-h total period; however, there was a slight overconsumption of calories during the dark phase for both concentrations. *Significant difference (P < 0.05) from saline infusions.

View larger version (22K): [in this window] [in a new window]   Fig. 4. Total caloric intakes (means ± SE) during the 20-h daily period (A and C) and the 12-h dark phase (B and D) for S129 mice (n = 7) and NT-4KO mice (n = 7) after glucose infusions. S129 mice showed a small overconsumption of calories during the 20-h total period for both concentrations, but only for the larger 25% concentration during the dark phase. NT-4KO mice did not show overconsumption for any of the infusates. *Significant difference (P < 0.05) from saline infusions.

Discussion

The present experiment established a number of conclusions about the use of a second macronutrient, glucose, in the yoked infusion paradigm that complements and extends the findings of experiments 1 and 2. First, the present experiment established two concentrations of glucose that produce a dose-dependent effect on pellet intake. In addition, these concentrations did not seem to cause nausea or malaise, as indicated by the normal cumulative intakes on glucose infusion days and the typical body weight gains. Finally, even though the lower concentration of glucose was occasionally ineffective, dose-dependent effects were observed for most meal-taking parameters. Importantly, the results suggest that NT-4KO mice were as sensitive to the carbohydrate infusions as were their S129 controls, indicating that the insensitivities of the NT-4KOs to Intralipid reported in experiment 2 may be specific to fats.

Earlier studies have indicated that fat and glucose have differential effects on satiation and satiety (2, 29). Such differences also appear in the present yoked infusion paradigm if one compares the intrameal and intermeal results of the present experiment with those of the previous two experiments. Since the higher glucose concentration (25%) was calorically matched to the lower Intralipid concentration (10%), a direct comparison can be made between those two macronutrient infusions. C57BL/6 mice showed the same patterns of meal taking changes after glucose infusions as after Intralipid infusions, but the two nutrients produced different magnitudes of changes. For example, comparing equicaloric infusates (10% Intralipid and 25% glucose), the glucose infusions were detected more quickly (e.g., average meal duration after 25% glucose infusions decreased 19% compared with 8% for 10% Intralipid in the dark phase, P < 0.05), and similar trends were observed for meal size. However, intermeal effects were smaller after glucose infusions (e.g., average IMI after 25% glucose infusions increased 15% compared with 30% after 10% Intralipid infusions in the dark phase, P < 0.05). Therefore, in line with previous studies, glucose seems to have rapid intrameal satiating effects, yet much smaller intermeal satiety effects.

Because the range of caloric concentrations for the glucose infusions of the present experiment was shifted to the left with respect to the range of caloric concentrations of Intralipid in experiment 2 and because the older S129 animals had lower saline-infusion baseline intakes than did the younger C57BL/6 mice, it was more difficult to observe differences in intrameal effects for the S129 mice. This was likely a floor effect as average meal durations were small (e.g., 2.35 min during the dark phase) for S129 mice. Regardless, S129 mice still displayed intermeal effects which were much smaller with glucose infusions than with lipid infusions (e.g., average satiety ratio after 25% glucose increased 28% compared with 41% after 10% Intralipid infusions in the dark phase, P < 0.05) and consistently showed dose-dependent effects.

In the present experiment, in contrast to the deficits in lipid feedback that NT-4KO mice evidenced in experiment 2, NT-4KO mice were as sensitive to IG glucose infusions as were their S129 controls, suggesting that the vagal deficits in the NT-4KO mice do not affect the animals' ability to reduce their pellet consumption when challenged with intake-yoked infusions of glucose. This could be due to a number of possible mechanisms. Perhaps most likely, simple carbohydrates, particularly glucose, are absorbed more rapidly than fat, and could have therefore reached the circulation and thus systemic and central receptors in the amount of time observed. Infused glucose rapidly reaching the circulation might raise blood glucose levels and cancel any glucoprivic signals that might otherwise play either permissive or more direct roles in influencing the feeding of the animals (also see Ref. 26). This early uptake of the carbohydrate might diminish the need for the vagus to relay preabsorptive glucose signals to the brainstem, unlike the situation for infusions of fat.

	GENERAL DISCUSSION

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The present experiments were designed to evaluate whether the NT-4KO mouse, with its reduction of vagal innervation of the intestines, has corresponding losses in sensitivity to dietary macronutrients, particularly lipids, infused into the GI tract. The question was addressed with a test paradigm that yoked small, discrete infusions of macronutrient solutions to the ad libitum self-paced feeding of mice in their home cages and that used meal pattern analyses to assess the detailed effects of the infusions.

Both the results comparing the feeding of NT-4KO mice and their controls as they received macronutrient infusions in the yoked infusion paradigm and several observations on the utility of the test paradigm need to be examined in additional detail.

Deficits in the NT-4KO Mouse

Previous studies examining the neural deficits of NT-4 deletion (7) have discovered a significant loss of vagal afferent neurons and a corresponding loss of intestinal vagal mechanoreceptors. In addition, there was a significant loss of vagal fibers passing through the muscle layers, indicating that the innervation to the mucosal layer was also likely to be compromised. Fox et al. (7) also found that, on an ad libitum diet without infusions, NT-4KO mice showed some loss in the ability to detect nutrients as indicated by changes in meal parameters, such as an increase in meal size and duration.

In view of such observations, we initially focused on the sensitivity of NT-4KOs to lipid infusions into the GI tract. We hypothesized that the KO mice would evidence a loss of sensitivity in the detection of such infusions.

Intralipid Infusions: NT-4KOs were Less Sensitive to Lipids Infused Into the GI Tract

In experiment 2, a loss of sensitivity to Intralipid infusions was observed for the NT-4KO mice. A number of parameters were indicative of a lack of nutrient sensing or detection after fat infusions, including smaller decreases in meal size and duration, and smaller increases in IMI and satiety ratio. In addition, on days when fat was infused, NT-4KO mice significantly overconsumed in terms of total calories, indicating they did not compensate for the calories from the fat infused by reducing their pellet intake, as the C57BL/6 and S129 control strains did.

Although it is not practical to perform a formal dose response analysis with only two doses of a compound, it may be instructive in terms of designing future experiments to note that for a number of the different meal pattern parameters measured, the data of the NT-4KOs were consistent with the working hypothesis that the dose response curve for intestinal infusions was shifted to the right. Certainly, at the higher dose of Intralipid, some residual sensitivity was detected in the NT-4KOs.

Some of the deficits in responding to Intralipid infusions, specifically the deficits detected by the intrameal measures of meal size and meal duration, appeared so rapidly following meal onsets that they were presumably preabsorptive effects. The most obvious explanations for these effects would be the loss of the vagal chemoreceptors that are located in the mucosa and that have been implicated both in sensing lipids directly (14, 23), as well as in responding to the release of local paracrine factors (10, 19, 25).

Other deficits in responding to Intralipid infusions, specifically those losses detected in the intermeal measures of satiety ratio and IMI, as well as those deficits detected in the longer-term or 20-h measures of compensatory adjustments in pellet intake, point to the conclusion that the NT-4KO animals also have some loss in their ability to make long-term compensations for fat calories. Such deficits occurring well after the lipids have been absorbed might be explained by several hypothetical mechanisms (e.g., the vagal projections to the liver have never been examined in the NT-4KO, and they could also be affected by the mutation), but a particularly parsimonious explanation might be simply that preabsorptive sensing of lipids plays a critical role in the overall regulation of fats and that compensations for the loss of such key mechanisms are incomplete.

NT-4KOs are as Sensitive as Controls to Glucose Infusions

Unlike with Intralipid infusions, NT-4KO mice that received yoked infusions of glucose had similar changes to meal parameters as the S129 mice. Such nutrient selectivity would reinforce the conclusion that the deficits resulted from the loss of mucosal chemoreceptors, which would have some chemical specificity, rather than the correlated losses of intestinal mechanoreceptors, which presumably would have been stimulated in at least relatively similar fashions by both lipid and glucose infusions.

It should be noted, however, that it may be premature to assume that NT-4KO animals would respond normally or similar to controls to all carbohydrates. Conceivably, if the yoked infusion paradigm were used with complex carbohydrates that would require digestion and would remain longer in the GI tract were used as the nutrient infusates, the NT-4KO model might evidence an impairment. Such an hypothesis would generally be consistent with the evidence that vagal afferents innervating the intestine have been shown to respond to sugars (20, 22).

Although we have provisionally concluded that the different responses of the NT-4KOs compared with those of their controls to the infused lipid and carbohydrate solutions are a reflection of the two genotypes' GI sensitivities to the two macronutrient classes, not all chemical dimensions of the solutions were matched or otherwise controlled in the present experiments. For example, the different solutions were not equivalent in their osmolarities (the 2 concentrations of Intralipid had osmolarities of, respectively, 150 and 300 mOsm/l, whereas the 2 glucose solutions had values of, respectively, 750 and 1,500 mOsm/l). Future comparisons certainly need to evaluate the additional chemical dimensions of the nutrient solutions. In a similar vein, additional experiments should investigate whether the neural losses of the NT-4KO translate into deficits in protein sensing or not. However, regardless of such future assessments of the precise chemical and nutrient specificities of the effects we have observed (and, for that matter, regardless of precisely what chemical signals of the different macronutrients are recognized in the GI tract), the present results clearly indicate a selective loss of sensitivity correlated with the loss of vagal afferents in the intestines of the NT-4KO mouse.

Lack of Caloric Compensation for Daily Trials Did Not Lead to Weight Gain

In experiment 2, the NT-4KO mice appeared to fall into positive energy balance on Intralipid infusion days. Nonetheless, over the 30-day experiments, the overall changes in body weights of the NT-4KOs and S129 controls were almost identical (P > 0.05). There are a number of reasons why the body weights may not have differed between the strains. Fat infusions only occurred every 2–3 days, and not everyday. Subsequently, for the majority of test days the animals were infused with saline as they consumed their maintenance diet. Had the fat infusions occurred daily, the body weights may have reflected the consistent failure to adequately reduce pellet intake to compensate for the fat calories infused; however, this is a possibility that needs to be addressed experimentally.

The Utility of the Yoked Infusion Paradigm

A number of advantages of the yoked infusion paradigm become evident in these experiments. First, the animal was allowed to remain in its home environment and was given no cues (e.g., moved to a different cage, being handled) that it was going to be tested. Second, utilizing meal analysis allowed for the distinction between intrameal and intermeal effects. As with any paradigm, however, there were drawbacks to the protocol. For instance, as a result of the animals being constantly connected to the apparatus, daily body weights were not feasibly obtained. Body weights were measured, however, at the beginning and end of the experiment (at day 0 and day 31) to compare any changes that occurred. In addition, body weights were taken daily before the start of the experiment.

	APPENDIX A

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Justification of Meal Parameter Criteria for NT-4KO Mice

The criteria for meal initiations (3 pellets in a 7-min time period) and terminations (10 min of no eating) were previously evaluated and verified using C57BL/6 mice (3). To verify these criteria in a mutant strain of mice, the same assessment procedure was repeated using NT-4KO mice. For each of two mice, the number of putative meals was calculated using a range of criterion values for the start of a meal (from 1 to 10 pellets in a time period of 5 to 20 min).

Based on these changes, the number of meals distinguished was then plotted as a function of the different criterion values for the start of a meal, and the resulting graphs were evaluated to identify the most stable regions. The maximum stability was taken as that point in the graphed function with the minimum slope, such that a change in criterion by one pellet or 1 min did not drastically change the resulting number of meals. Low stability was defined as an area where a change in a single pellet or minute as the criterion for the start of a meal resulted in a larger change in meal number.

The most stable regions occurred between two and five pellets as the criterion for the start of a meal, similar to the result previously reported for the C57BL/6 mice. In addition, the time parameter contributed little to the differences in resulting meal number, as it did for the C57BL/6 mice, and the range of 5 to 20 min did not alter the meal number in the majority of cases. The end of a meal has previously shown to also play less of a factor; therefore 10 min of no feeding was chosen as the end of meal as it was within the range of values indicated and typically used in meal patterning studies.

	APPENDIX B

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Changes in Caloric Tracking of Pellets Following Macronutrient Infusions

Since animals are able to learn to track changes in the caloric values of foods in some situations (12, 30) and since the net effect of intragastric caloric infusions might be tantamount to increasing the caloric content of the pellets, a check for alterations in baseline consumption of pellets after lipid (experiments 1 and 2) and glucose (experiment 3) infusion trials was performed. This additional analysis consisted of comparing all meal parameters for the first and second days following a fat infusion, as well as the days before and after the infusions, which would indicate whether there were any significant changes in the animals' expectations of the postingestive consequences of the pellets. Values for each meal parameter were averaged for each animal for each concentration of infusion and then averaged for all animals. If the animals had "learned" during infusion days that each pellet was associated with a larger caloric content, corresponding changes in meal parameters should have occurred on subsequent days when saline was infused.

In experiment 1, some parameters showed nonsignificant trends on days following fat infusions in C57BL/6 mice, but the differences were not consistent across meals, and parameters that usually change together (e.g., meal duration and size) did not. Similarly, the days before and after fat infusions were also compared, and a small number of parameters showed nonsignificant trends but were not consistent across meals. Importantly, parameters that normally change together did not, supporting the idea that these trends were not indicative of an overall change concerning the pellets.

In experiment 2, a two-way ANOVA revealed an effect of day for the third meal IMI (P = 0.03), however, the rest of the parameters did not differ. Similarly, the days before and after fat infusions were also compared. Again, an effect of day was observed for a single parameter (first meal satiety ratio, P = 0.01), but overall the parameters were similar, and changes that normally occur together did not.

In experiment 3, C57BL/6 mice showed no significant changes in the first and second days after glucose infusions of the first three individual meals, the dark phase, or 20-h daily means. Similarly, there were no differences between the first and second days after glucose infusions for the S129 or NT-4KO mice for any of the time periods. In addition, the days before and after glucose infusions for C57BL/6 mice were similar for all of the time periods examined. For the S129 and NT-4KO mice, there was a slight difference between the days before and after glucose infusions for the first meal size (P = 0.046), but this was not observed for the second or third meals, or for the 20-h daily or dark-phase periods.

	GRANT

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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-27627.

	ACKNOWLEDGMENTS

 
We thank Drs. E. Fox, K. Kinzig, and R. Shi for comments on earlier drafts of this manuscript. We also thank Dr. R. Phillips for assistance with the development of the mouse surgery, as well as J. Lui, K. Fugo, and M. Grubbs for assistance with data collection and analysis.

This report is based on a thesis submitted by M. Chi in partial fulfillment of the requirements of the degree of Ph.D. at Purdue University, and a preliminary report by M. M. Chi and T. L. Powley of a portion of this work appeared in abstract form at the 35th annual meeting of the Society for Neuroscience, 2005.

	FOOTNOTES

 

Address for reprint requests and other correspondence: M. M. Chi, Dept. of Cell and Molecular Physiology, Univ. of North Carolina at Chapel Hill, 103 Mason Farm Rd., MBRB, CB #7545, Chapel Hill, NC 27599-7545 (e-mail: mchi{at}med.unc.edu)

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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