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1 Monell Chemical Senses Center and 2 University of Pennsylvania, Department of Biochemistry & Biophysics, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Administration of the fructose analog 2,5-anhydro-D-mannitol (2,5-AM) stimulates eating in rats fed a low-fat diet but not in those fed a high-fat diet that enhances fatty acid oxidation. The eating response to 2,5-AM treatment is apparently triggered by a decrease in liver ATP content. To assess whether feeding a high-fat diet prevents the eating response to 2,5-AM by attenuating the decrease in liver ATP, we examined the effects of the analog on food intake, liver ATP content, and hepatic phosphate metabolism [using in vivo 31P-NMR spectroscopy (NMRS)]. Injection (intraperitoneal) of 300 mg/kg 2,5-AM increased food intake in rats fed a high-carbohydrate/low-fat diet, but not in those fed high-fat/low-carbohydrate (HF/LC) food. Liver ATP content decreased in all rats given 2,5-AM compared with saline, but it decreased about half as much in rats fed the HF/LC diet. NMRS on livers of anesthetized rats indicated that feeding the HF/LC diet attenuates the effects of 2,5-AM on liver ATP by reducing phosphate trapping. These results suggest that rats consuming a high-fat diet do not increase food intake after injection of 2,5-AM, because the analog is not sufficiently phosphorylated and therefore fails to decrease liver energy status below a level that generates a signal to eat.
feeding behavior; fuel metabolism; nuclear magnetic resonance
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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EVIDENCE INDICATES THAT FEEDING behavior is controlled, in part, by changes in hepatic energy metabolism, which generate neural signals that are carried from the liver to the central nervous system by vagal afferent neurons (3, 11). Although the precise nature of the hepatic metabolic events that trigger this signal is unknown, accumulating evidence implicates changes in liver ATP content or some other aspect of liver energy status as the stimulus. Much, but not all (e.g., 7), of this evidence has been derived from studies of the eating response that occurs after administration of the fructose analog 2,5-anhydro-D-mannitol (2,5-AM), which acts in the liver to stimulate feeding behavior (19).
Similar to fructose, 2,5-AM is phosphorylated in the liver at the one and six positions, but unlike fructose, it is not metabolized further (16). As a result, inorganic phosphate is trapped in these phosphorylated forms of 2,5-AM, thereby limiting the availability of inorganic phosphate for ATP synthesis, which, in turn, causes liver ATP to decline. Giving rats 2,5-AM either by injection or gavage stimulates food consumption during the daylight hours when these nocturnal animals normally eat very little (18). This increase in feeding behavior occurs within the time frame of the decrease in liver ATP (10, 13). More importantly, the reduction of liver ATP precedes the eating response as would be expected if it were acting as a stimulus (10). In addition, if access to food is delayed after treatment with 2,5-AM, rats will increase food intake as long as ATP remains low, but they show little or no eating response if time is allowed for ATP content to recover (10). Consistent with a phosphate-trapping mechanism of action, administration of exogenous phosphate prevents both the drop in ATP and the stimulation of feeding behavior produced by 2,5-AM treatment (14).
The eating response to administration of 2,5-AM is modulated by changes in fat fuel oxidation. Pharmacological inhibition of fatty acid oxidation synergistically potentiates the eating response to 2,5-AM (4, 8, 15), and combined treatment with 2,5-AM and a fatty acid oxidation inhibitor also reduces liver energy status in a synergistic fashion (8), which further points to a role of hepatic energy status in stimulating food intake. In contrast, rats maintained on a high-fat diet that facilitates fat oxidation do not increase food intake after 2,5-AM injection as do rats fed a low-fat diet (6, 15). It is not known whether feeding a high-fat diet also attenuates the effect of 2,5-AM on liver ATP content. The present experiments were designed to address this question and examine the mechanisms involved.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Animals. Male Sprague-Dawley CD rats (Charles River, Wilmington, MA) weighing 300-400 g at the beginning of the experiments were used. Rats were maintained on a 12:12-h light-dark cycle and housed individually with food and water available ad libitum unless noted otherwise. Animals were fed either a high-carbohydrate/low-fat (HC/LF) or high-fat/low-carbohydrate (HF/LC) diet, which was custom-made (ICN Biochemicals, Aurora, OH or Dyets, Bethlehem, PA) and of equal caloric density (3.2 kcal/g) (12). The HC/LF diet contained (by calories) 63% carbohydrate, 13% fat, and 24% protein, whereas the HF/LC diet contained 63% fat, 13% carbohydrate, and 24% protein. Rats were fed a given diet for at least 2 wk before being used for experiments. Experimental protocols were approved by the Institutional Animal Care and Use Committee of the Monell Chemical Senses Center.
Food intake and liver ATP. Rats in each diet group were further divided into two groups (n = 9-10), which were injected (intraperitoneal) with either isotonic saline or 300 mg/kg 2,5-AM in saline (2 ml/kg) ~2 h after the start of the daylight period. Food intakes (to the nearest 0.1 g corrected for spillage) were measured 1, 2, and 4 h after injections. One week later, rats were reinjected at the same time with vehicle or 2,5-AM, deprived of food, and anesthetized (100 mg/kg Ketamine with 1 mg/kg ip acepromazine) 30 min later for collection of liver tissue.
In vivo 31P-NMR. An intraperitoneal catheter was implanted 6-7 days before NMR experiments. Rats fed either the HC/LF or HF/LC diet (n = 6/group) were anesthetized as above, and a piece of Silastic tubing (0.025 × 0.047, internal diameter × outer diameter) was inserted through a small incision in the lower abdominal muscle. A collar made of Silastic tubing, which was fitted over the catheter, was placed on the interior of the abdominal wall and sutured to the muscle to secure the catheter in place. The other end of the catheter was connected to a larger piece of Silastic tubing, which was tunneled under the skin to between the scapulas where it was secured subcutaneously using Silastic mesh. The catheter was then filled with saline and plugged, and the incision between the scapulas was closed with sutures.
For NMR measurements, rats from each diet group were anesthetized as above, and the upper abdominal muscles over the liver were removed via a midline incision. The incision was closed with surgical glue, and a 15-mm (diameter) triple-tuned surface coil tuned to 121.5 MHz for 31P was positioned over the liver and sutured in place. The catheter between the scapulas was exteriorized and connected to a long piece of tubing for remote injection. Rats were then secured by skin sutures to a Plexiglas holder, which was inserted into the magnet. After animals were placed in the magnet, the field of homogeneity was adjusted using a proton signal from the liver. After collection of baseline spectra for 20 min, 300 mg/kg of 2,5-AM were injected as a bolus via the previously implanted intraperitoneal catheter, and spectra were collected for another 100 min. A Bruker AMX-300/SWB spectrometer (150-mm bore, 7-T vertical magnet) was used for 31P-NMR. The acquisition parameters for 31P spectra were: 250 free induction decays, 60° pulse, and 1.4-s recycle time. Spectra were smoothed by an exponential multiplication of 40 Hz. The peaks for ATP (using ATP-
),
phosphomonoesters (PME), Pi, and total phosphate (P) were
identified according to Desmoulin et al. (1), and values
were calculated from the area under the respective peak for each
constituent. Spectra were collected approximately every 10 min.
Data are presented as percent change from baseline.
Liver ATP assay.
The liver was exposed through a midline abdominal incision, a portion
of the median lobe was excised, immediately freeze-clamped using
aluminum blocks previously cooled in liquid nitrogen, and then immersed
in liquid nitrogen. Liver samples were stored at 
70°C before
extraction in which liver samples were pulverized under liquid nitrogen
and homogenized in 6% perchloric acid. Homogenates were centrifuged,
and the resulting supernatants were adjusted to pH 7.8 with 69%
K2CO3, placed on ice for 1 h, and
centrifuged again. Supernatants from these neutralized extracts were
diluted 1:200 with purified water and stored at 70°C before assay
for ATP. ATP was assayed enzymatically using a commercial kit (Sigma FL-AA, Technical Bulletin No. BAAB-1) based on the luciferin-luciferase reaction.
Statistical analyses. Data were analyzed by two-way ANOVA; time course data were analyzed by two-way ANOVA with repeated measures. Post hoc comparisons were made using a Newman-Keuls test when the main or interaction terms of the ANOVA were statistically significant at P < 0.05. Student's t-test was used for all other two-group comparisons. Data are reported as means ± SE.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Food intake and liver ATP.
Rats fed the HC/LF diet increased food intake by 400% during the
4 h after injection of 2,5-AM compared with saline (2.5 ± 0.3 vs. 0.6 ± 0.2 g), whereas rats fed the HF/LC diet showed
no significant change in intake [F(1,34) = 10.3, P < 0.0002 for diet × treatment
interaction; Fig. 1]. This differential
effect of diet on the eating response to 2,5-AM was seen at each time
point starting at 1 h [F(1,34) = 4.2, 11.7, and 15.0, P < 0.05, 0.002, and 0.001 at 1-, 2-, and 4-h time points, respectively, for diet × treatment
interaction].
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13 vs. 27%). Consequently, liver ATP levels in rats fed the HF/LC
diet and given 2,5-AM were significantly higher than those in
2,5-AM-treated rats fed the HC/LF diet
[t(15) = 2.4, P < 0.03]
and did not differ from those in saline-treated rats eating the HC/LF
diet. Rats fed the HF/LC diet had higher liver ATP content than those
fed the HC/LF food irrespective of whether they were given 2,5-AM or
saline [F(1,33) = 9.0, P < 0.01]. Because liver ATP levels were similar in rats from the two
diet groups that were injected with saline, the overall effect of diet
appears to be due primarily to the higher level of ATP in livers from
rats fed the HF/LC diet and injected with 2,5-AM.
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In vivo 31P-NMR.
Under the conditions of the current experiment, liver ATP, PME,
Pi, and total P showed no net change from baseline after
injection of isotonic saline (13; unpublished observations). Liver ATP
decreased significantly [F(10,100) = 45.2, P < 0.00001] from baseline by ~25% starting
10-20 min after injection of 2,5-AM, and it stabilized 50-60
min postinjection (Fig. 3). Although ATP
decreased similarly in the two diet groups over the entire 100-min
observation period following injection, liver ATP declined
significantly more in rats fed the HC/LF diet compared with the HF/LC
diet in the first 50 min after 2,5-AM treatment
[F(1,10) = 7.4, P < 0.025].
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Previous studies showed that injection of 2,5-AM stimulates food intake in rats fed a low-fat but not high-fat diet (6, 15). The present results confirm this observation and extend it to show that this effect of diet on the eating response to 2,5-AM is associated with differential changes in liver ATP levels and phosphate metabolism. These effects of diet on the hepatic metabolic response to 2,5-AM suggest a mechanism by which feeding a high-fat diet prevents the eating response to 2,5-AM and also provide further evidence that a decrease in liver energy status generates a signal that triggers feeding behavior.
Similar to fructose, 2,5-AM is phosphorylated at the one and six position in the liver, but, unlike fructose, it is not further metabolized via glycolytic pathways (16). Consequently, phosphate in liver is trapped in the 1-phosphate and 1,6-phosphate forms of 2,5-AM after administration of the analog (13). Several lines of evidence indicate that this phosphate trapping underlies both the decrease in liver energy status and the increase in food intake after 2,5-AM treatment. The drop in liver ATP after injection of 2,5-AM is accompanied by a substantial decrease in Pi (Fig. 3) (13) and a marked increase in PME (Fig. 3) (13), which are comprised largely of phosphorylated 2,5-AM (13). The eating response to 2,5-AM treatment is also accompanied by increasing phosphorylation of the analog in the liver, and it wanes as the amount of phosphorylated 2,5-AM in the liver declines, apparently as a result of dephosphorylation (19). In addition, administration of exogenous phosphate both reverses the reduction in liver ATP and suppresses the eating response induced by 2,5-AM injection (14).
The differential effects of diet composition on the hepatic metabolic and behavioral responses to 2,5-AM may also be due to differences in phosphate trapping in the liver. Measurements of 31P metabolism using NMR spectroscopy showed that, after intraperitoneal injection of 2,5-AM, liver ATP levels did not decline as much nor did liver PME and total P increase as much in rats fed the HF/LC diet as in those fed the HC/LF diet. Also, these effects on liver ATP, PME, and total P were seen primarily within the first 50 min after injection of 2,5-AM (Fig. 3), an interval in keeping with the time course of the eating response to intraperitoneal injection of the analog (Fig. 1) (10). These results suggest that feeding a high-fat diet ameliorates the effects of 2,5-AM on both liver energy status and feeding behavior by limiting phosphate trapping in the liver. Results from experiments (9) using isolated hepatocytes in vitro, which indicate that feeding rats the HF/LC diet decreases the oxidation of carbohydrates, are consistent with this interpretation.
Feeding rats the HF/LC diet prevented the eating response to 2,5-AM treatment, but it did not eliminate the effects on liver phosphate metabolism, including that on hepatic ATP content. This observation suggests that liver ATP must decline beyond some threshold level to trigger the eating response. Although the results might suggest that a decrease in liver ATP of more than 15-20% should stimulate feeding behavior, such a conclusion is not warranted at this time. First, it is not clear that changes in ATP content per se provide the signal that elicits eating; other parameters of liver energy status may be involved. Second, in experiments to date, liver measurements were taken either from one freeze-clamped lobe of the liver or from the whole liver using NMR spectroscopy. Therefore, it is unclear whether a drop in liver ATP of 15-20% reflects a decrease of that magnitude throughout the liver parenchyma or a much larger decrease in discrete areas of the liver or liver parenchyma. Further research is required to determine the nature, degree, and location of changes in liver energy status that stimulate food intake before quantitative estimates of a threshold can be made.
The effect of 2,5-AM on liver energy metabolism appears to generate a signal that is carried to the central nervous system by vagal sensory neurons (17). 2,5-AM treatment activates neurons in areas of the brain that receive input from the vagal afferent system (4-6, 17), and, parallel to its effects on food intake, it does so in rats fed a HC/LF but not HF/LC diet. The lack of a behavioral response in rats fed the HF/LC diet does not appear to be due to an inability to increase food intake, because administration of the fatty acid oxidation inhibitor methyl palmoxirate stimulates eating in rats fed this diet (2), and coinjection of 2,5-AM potentiates this response (8, 15). It also seems unlikely that the dietary manipulation prevents the neuronal and behavioral responses to 2,5-AM by a direct effect in the brain, because all evidence points to an hepatic mechanism of action for 2,5-AM and a decrease in liver energy status as the stimulus to eat (10, 13-15, 19). In addition, as shown in the following paper (9), 2,5-AM is less effective in decreasing liver energy status in isolated hepatocytes from rats fed the HF/LC diet compared with those from animals eating the HC/LF food. The experiments reported here and in the companion paper suggest that rats fed a high-fat diet lack a neuronal and behavioral response to 2,5-AM treatment, because the analog produces a drop in liver energy status that is insufficient to generate a neural signal from the liver.
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ACKNOWLEDGEMENTS |
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We thank T. Herman and J. Nuss for technical assistance and Dr. K. Torii for the generous donation of 2,5-anhydro-D-mannitol.
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FOOTNOTES |
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This work was supported by National Institutes of Health Grant DK-53109.
Address for reprint requests and other correspondence: M. I. Friedman, Monell Chemical Senses Center, 3500 Market St., Philadelphia, PA 19104 (E-mail: friedman{at}monell.org).
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.
10.1152/ajpregu.00156.2001
Received 15 March 2001; accepted in final form 30 October 2001.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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1.
Desmoulin, F,
Cozzone PJ,
and
Canioni P.
Phosphorus-31 nuclear-magnetic-resonance study of phosphorylated metabolites compartmentation, intracellular pH and phosphorylation state during normoxia, hypoxia and ethanol perfusion, in the perfused rat liver.
Eur J Biochem
162:
151-159,
1987[ISI][Medline].
2.
Friedman, MI,
Harris RB,
Ji H,
Ramirez I,
and
Tordoff MG.
Fatty acid oxidation affects food intake by altering hepatic energy status.
Am J Physiol Regulatory Integrative Comp Physiol
276:
R1046-R1053,
1999
3.
Friedman, MI,
Rawson NE,
and
Tordoff MG.
Hepatic signals for control of food intake.
In: The Genetics and Molecular Biology of Obesity, edited by Bray GA,
and Ryan DH.. Baton Rouge, LA: LSU, 1996, p. 318-339.
4.
Horn, CC,
Aleymayehu A,
and
Friedman MI.
Neural substrate for an integrated metabolic control of feeding behavior.
Am J Physiol Regulatory Integrative Comp Physiol
276:
R113-R119,
1999
5.
Horn, CC,
and
Friedman MI.
2,5-Anhydro-D-mannitol induces Fos-like immunoreactivity in hindbrain and forebrain: relationship to eating behavior.
Brain Res
779:
17-25,
1998[ISI][Medline].
6.
Horn, CC,
and
Friedman MI.
Metabolic inhibition increases feeding and brain Fos-like immunoreactivity as a function of diet.
Am J Physiol Regulatory Integrative Comp Physiol
275:
R448-R459,
1998
7.
Ji, H,
and
Friedman MI.
Compensatory hyperphagia after fasting tracks recovery of liver energy status.
Physiol Behav
68:
181-186,
1999[Medline].
8.
Ji, H,
Graczyk-Milbrandt G,
and
Friedman MI.
Metabolic inhibitors synergistically decrease hepatic energy status and increase food intake.
Am J Physiol Regulatory Integrative Comp Physiol
278:
R1579-R1582,
2000
9.
Ji, H,
Graczyk-Milbrandt G,
Osbakken MD,
and
Friedman MI.
Interactions of dietary fat and 2,5-anhydro-D-mannitol on energy metabolism in isolated rat hepatocytes.
Am J Physiol Regulatory Integrative Comp Physiol
282:
R715-R720,
2002
10.
Koch, JE,
Ji H,
Osbakken MD,
and
Friedman MI.
Temporal relationships between eating behavior and liver adenine nucleotides in rats treated with 2,5-AM.
Am J Physiol Regulatory Integrative Comp Physiol
274:
R610-R617,
1998
11.
Langhans, W.
Role of the hepatic afferent nerves in the control of food intake.
In: Liver and Nervous System, edited by Häussinger D,
and Jungermann K. Dordrecht. The Netherlands: Kluwer Academic, 1998, p. 196-208.
12.
Ramirez, I,
and
Friedman MI.
Dietary hyperphagia in rats: role of fat, carbohydrate, and energy content.
Physiol Behav
47:
1157-1163,
1990[Medline].
13.
Rawson, NE,
Blum H,
Osbakken MD,
and
Friedman MI.
Hepatic phosphate trapping, decreased ATP, and increased feeding after 2,5-anhydro-D-mannitol.
Am J Physiol Regulatory Integrative Comp Physiol
266:
R112-R117,
1994
14.
Rawson, NE,
and
Friedman MI.
Phosphate loading prevents the decrease in ATP and increase in food intake produced by 2,5-anhydro-D-mannitol.
Am J Physiol Regulatory Integrative Comp Physiol
266:
R1792-R1796,
1994
15.
Rawson, NE,
Ulrich PM,
and
Friedman MI.
Fatty acid oxidation modulates the eating response to the fructose analogue 2,5-anhydro-D-mannitol.
Am J Physiol Regulatory Integrative Comp Physiol
271:
R144-R148,
1996
16.
Riquelme, PT,
Wernette-Hammond ME,
Kneer NM,
and
Lardy HA.
Mechanism of action of 2,5-anhydro-D-mannitol in hepatocytes.
J Biol Chem
259:
5115-5123,
1984
17.
Ritter, S,
Dihn TT,
and
Friedman MI.
Induction of Fos-like immunoreactivity (Fos-li) and stimulation of feeding by 2,5-anhydro-D-mannitol (2,5-AM) require the vagus nerve.
Brain Res
646:
53-64,
1994[ISI][Medline].
18.
Tordoff, MG,
Rafka R,
DiNovi MJ,
and
Friedman MI.
2,5-Anhydro-D-mannitol: a fructose analogue that increases food intake in rats.
Am J Physiol Regulatory Integrative Comp Physiol
254:
R150-R153,
1988
19.
Tordoff, MG,
Rawson N,
and
Friedman MI.
2,5-Anhydro-D-mannitol acts in liver to initiate feeding.
Am J Physiol Regulatory Integrative Comp Physiol
261:
R283-R288,
1991
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