Choice of Lard, But Not Total Lard Calories, Damps Adrenocorticotropin Responses to Restraint
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内分泌学杂志 2005年第5期
Department of Physiology, University of California San Francisco, San Francisco, California 94143-0444
Address all correspondence and requests for reprints to: Mary F. Dallman, Department of Physiology, University of California San Francisco, San Francisco, California 94143-0444. E-mail: dallman@itsa.ucsf.edu.
Abstract
Although rats given the choice of eating high-density calories as concentrated sucrose solutions or lard exhibit reduced responsivity in the hypothalamo-pituitary-adrenal axis, rats fed high-fat diets have normal or augmented responses to stressors. To resolve this apparent discrepancy, we compared in adult male rats the effects of 7-d feeding with lard + chow (choice) to feeding a 50% lard-chow mixture (no-choice) and to chow only. Rats with choice composed diets with 50–60% total calories from lard. Rats were exposed to 30 min of restraint on d 7. In the choice group, there was a robust inhibition of ACTH and corticosterone responses to restraint compared with chow or no-choice groups. Total caloric intake was less with choice than no-choice. Fat depot weights and body weight gain were similar in the high-fat groups. Leptin concentrations were equal but insulin was higher in the choice group. We conclude the following: 1) choice of eating high-density calories strongly damps hypothalamo-pituitary-adrenal responses to stress; without choice, high-density diet is ineffective; and 2) insulin may signal metabolic well-being, and may act through hypothalamic sites to reduce caloric intake but through forebrain sites to damp stress responses.
Introduction
IN RECENT YEARS we have provided either separate containers of 30% sucrose or lard, or both, in addition to standard chow, after adrenalectomy (1, 2, 3, 4, 5), and after chronic stressors in intact or adrenalectomized rats (1, 6, 7). In general, the provision of these "comfort" foods reduced activity in the hypothalamo-pituitary-adrenal (HPA) axis, and rats that ate the comfort foods increased mesenteric fat stores. These results led us to propose that an unidentified metabolic feedback signal (8) from central fat stores modulates the HPA response to stress (9).
Although rats that voluntarily compose the percentage of lard in their diets have reduced HPA responses, there is a considerable literature that suggests that a high-fat diet stimulates activity in the HPA axis (10, 11, 12, 13, 14, 15, 16). Moreover, lipid acutely infused into rats also stimulates the HPA axis (17, 18, 19). Experimental resolution of these differences in the reported effects of high-fat feeding on function in the HPA axis is important.
The most obvious difference between our experiments showing inhibition, and those of others showing stimulation of HPA activity is choice or no choice of the rats to eat fat. If such differences can be shown within an experiment, the differences between fat-fed groups might reveal a signal that acts to inhibit activity in the HPA axis. Moreover, in an unpublished experiment (K. D. Laugero and F. Gomez, unpublished data), when we removed choice of drinking sucrose by adding 30% sucrose to the saline solution of adrenalectomized rats, this stimulated, and did not inhibit ACTH as voluntary sucrose ingestion does. Because rats develop a salt appetite after adrenalectomy (20), they drank large amounts of the saline-sucrose mix to obtain salt. Thus, the component of choice seemed as though it might be quite important in the differences observed after various regimens involved in feeding rats a high caloric nutrient.
The current experiments were performed to distinguish between the effects of voluntary and involuntary lard intake for 7 d before intact rats were stressed with an acute 30-min period of restraint. Caloric intake, timing of intake, metabolic hormone concentrations, and fat depot weights were measured as well as the acute ACTH and corticosterone responses to restraint.
Materials and Methods
Animals
Male Sprague Dawley rats, 60 d old, weighing 280–320 g from Bantin and Kingman Suppliers (Freemont, CA) (experiment 1a and 1b) and Simonsen Laboratories, Inc. (Gilroy, CA) (experiment 2) were housed in University of California San Francisco (UCSF) Lab Animal Resources Center in temperature- (21–23 C) and light- (12 h light; lights on at 0700 h) controlled rooms. Rats were housed individually in hanging wire cages and given food (Purina Chow no. 5008; Purina, St. Louis, MO) and water ad libitum until the start of the experiment, at least 4 d after arrival at UCSF. All procedures were approved by the UCSF Institutional Animal Care and Use Committee.
Experimental design
On d 0 at the end of the light cycle (at 1800 h) until d 7, each group was provided with one diet; the experiments differed in the specific array of diets offered. The diets were: a cup of lard (Armour, 9 cal/g) in addition to Purina rodent chow (3.31 cal/g) (choice); a similar cup but with pelleted chow (chow); a moderate-fat diet containing 20% calories from lard and 80% calories from Purina chow (no-choice20%, NC20%; calculated energy content of 4.898 cal/g); or a high-fat diet containing 50% calories from lard and 50% calories from Purina Chow (no-choice50%, NC50%; calculated energy content of 6.155 cal/g). Body weight and food intake was recorded daily in the morning. On d 7 at 0900 h, each rat was removed from his home cage and within 2 min a blood sample was collected from a cut (1 mm) made across the tip of the tail. Rats were placed in restraint tubes for 30 min, were then removed from the tubes and replaced in their home cages. Blood was collected 15, 30, 60, 90, and 120 min after the initial tail nick (0 min). Blood (300 μl) was collected into heparinized Natelson blood collection tubes and kept at 4 C until centrifuged in the cold. Aliquots of plasma were stored at –20 C until assays. All rats were killed 4 h after start of the restraint stress (at 1300 h) by decapitation within 10 sec after they had been taken from their home cages. Trunk blood was collected in tubes with EDTA, kept on ice, and centrifuged in the cold. Aliquots of plasma were stored at –20 C until assayed. Individual mesenteric, epididymal, sc (inguinal), and perirenal white adipose tissues, thymus, and adrenals were dissected, cleaned, and weighed.
Experiment 1a: choice (n = 7), chow (n = 7); experiment 1b: choice (n = 7), chow (n = 6), no-choice20% (n = 7), no-choice50% (n = 7); experiment 2: choice (n = 5), chow (n = 5), no-choice50% (n = 8). The 50% lard- and 20% lard-diets were prepared fresh daily by pulverizing chow pellets, adding the calculated caloric proportion of lard and thoroughly mixing.
Plasma assays
Plasma glucose and triglyceride concentrations were measured in plate assays (5). All plasma hormone concentrations were determined by radioimmunoassay (5).
Statistical analyses
Data were first analyzed by two-way ANOVA. A significant (P 0.05) global effect of ANOVA was followed by one-way ANOVA and post hoc tests of individual group differences [Fisher’s projected least significant difference (PLSD)]. Corticosterone (B) and ACTH data were analyzed by ANOVA corrected for repeated measures over time. Rats were excluded from analysis in experiments 1a (n = 7), 1b (n = 5), and 2 (n = 3) because of missing blood samples (n = 10), food hopper problems (n = 1) or growth failure (n = 2). In addition, two choice rats were excluded because they had very high insulin concentrations (>6 ng/ml) and high basal ACTH as well as ACTH responses to restraint. The final numbers in each group are indicated in the figures.
Results
Experiment 1
Choice groups ate 50% of total calories from lard. To control for this amount of dietary lard, we provided one group of rats in experiment 1b with a mixture of 50% calories from lard with 50% calories from chow to establish a pair-fed group (no-choice50%). A second group was given a lard-chow diet with fewer calories (20%) from lard (no-choice20%) similar to that in a previous study of no-choice (10).
Both the chow and the choice groups in experiments 1a and 1b had similar caloric intake, body weight gain, and hormonal responses to the 30-min restraint. Therefore, we combined the results (Fig. 1).
FIG. 1. Rats with choice of lard intake eat fewer total calories but ingest 50% of those calories as lard. A, Caloric intake during the experiment (groups were provided chow (n = 9), 20% lard-chow mixture (no-choice20%, NC20%; n = 7), choice of chow + lard (n = 9), and 50% lard-chow mixture (no-choice50%, NC50%; n = 6); differences among groups are indicated by different letters). B, Composition of intake of lard as a percentage of the total in the choice group.
Groups on choice, as well as those on the no-choice20% and no-choice50% were hyperphagic compared with the chow group (Fig. 1A). The no-choice20% and no-choice50% groups had greater caloric intake than the choice group. Voluntary lard intake (expressed as percentage of total intake) was stable during the course of the experiment (Fig. 1B). Body weight gain over the 7-d period did not differ among groups (Table 1; ANOVA: F (3, 26) = 0.48; P = 0.7); however, relative fat stores and plasma leptin concentrations were significantly increased in the choice and no-choice50% groups, but not in the no-choice20% group (Fig. 2, A and B). Plasma insulin concentrations were increased in the choice compared with those in the chow, no-choice20% and no-choice50% groups (Fig. 2C). Plasma glucose and triglyceride concentrations were not different among the groups, although plasma free fatty acid concentrations tended to be modestly elevated in the choice and no-choice50% groups compared with the chow group (Table 1; P < 0.1).
TABLE 1. Experiment 1, a and b
FIG. 2. Choice increases fat depots, leptin, and insulin. A, Mesenteric and sc fat depot weights. B, Plasma leptin concentrations. C, Plasma insulin concentrations in groups eating chow (n = 9), medium (20% lard) diet (no-choice20%, NC20%; n = 7), choice (50% lard, 50% chow; n = 9), and high (no-choice50%, NC50%; n = 6) diet. Differences among groups are indicated by different letters above the bars (post hoc PLSD). Note the elevated insulin in the choice group.
Basal plasma ACTH concentrations were similar in all groups (Fig. 3A and Table 1) and exposure to restraint also increased plasma ACTH concentrations significantly. However, the ACTH response to stress was significantly decreased in choice compared with no-choice50% or no-choice20% groups (Fig. 3A). Initial corticosterone concentrations were slightly increased in choice and no-choice50% groups compared with those on chow (Fig. 3B and Table 1). Acute restraint increased plasma corticosterone concentrations significantly in all groups of rats. However, again, the corticosterone response to stress was significantly decreased in the choice compared with chow, no-choice20% and no-choice50% groups.
FIG. 3. Choice attenuates ACTH and corticosterone responses to restraint. A, ACTH responses to restraint (inset, area under the response curve). B, Corticosterone responses to restraint (inset, area under the response curve). Differences among groups are indicated by different letters above the bars (post hoc PLSD); number per group indicated on legend.
Experiment 2
To analyze further the differences between choice and no-choice50% groups, we repeated the experiment and included more food intake measurements to test whether rats with choice consumed their lard at different times of day from the no-choice50% group.
Rats with choice consumed 64 ± 3% of their calories from lard and were hyperphagic compared with the chow group. However, rats with no-choice50% had higher caloric intake compared with the choice group (Fig. 4A). Chow and no-choice50% groups consumed similar percentages of their total intake of calories during the light period, the first half and the second half of the dark period (Fig. 4, B and C). The choice group was similar in timing of intake to the other groups; however, the variance was larger (Fig. 4, B and C).
FIG. 4. Choice decreases overall caloric consumption but does not affect time of day that intake occurs. A, Caloric intake over the 7-d experiment is increased with lard, but less with choice than no-choice. B and C, The times of day in which calories are ingested (as a percentage of total intake) does not depend upon choice of lard intake. Because there was higher variance in the choice than nonchoice groups on caloric intake in B and C, the individual rat numbers are indicated in the choice group for each time of day. Chow, n = 4; choice of chow and lard, n = 6; no choice of chow and lard, n = 5.
ACTH and corticosterone responses to 30-min restraint are shown in Fig. 5, A and B, and the results confirmed the first experiment showing diminished ACTH responses to restraint in the choice group (Fig. 5, A and B). However, the corticosterone response in the choice group was significantly decreased only in the no-choice50% group, and was not different from the chow group in this experiment.
FIG. 5. Choice attenuates ACTH and corticosterone responses to restraint. A, ACTH responses to restraint (inset, area under the response curve). B, Corticosterone responses to restraint (inset, area under the response curve). Differences among groups are indicated by different letters above the bars in the inset (post hoc PLSD); number per group indicated on legend.
Body weight gain did not differ among the groups (Table 2). Relative fat stores and plasma leptin concentrations were increased in both choice and no-choice50% groups (Table 2). Plasma insulin concentrations were increased in the choice compared with those in the chow and no-choice50% groups (Table 2) and plasma adiponectin concentrations increased in the choice and increased slightly with no-choice50% diet (Table 2).
TABLE 2. Results from experiment 2
Discussion
Rats that ate a 50% lard-chow diet had normal HPA responses to acute restraint, whereas those selecting equal proportions of lard and chow had robustly diminished ACTH, and to a lesser extent, corticosterone responses. In the groups of rats voluntarily or involuntarily eating the high-fat diets, sc and intra-abdominal fat depot weights increased, to an equal extent compared with rats eating only chow. Additionally, there was no differential increase in body weight between the groups eating 50% lard mixed with chow or self-selecting the same percentages of lard and chow. Rats eating the lard-chow mixture had consistently increased caloric intake over the 7 d of study compared with the rats with choice. Obtained within experiments, these results confirm the apparently conflicting reports that high-fat diets stimulate HPA responses and that choice of high density calories diminishes HPA responses (see introduction).
We considered the possibility that voluntary lard ingestion might occur at different times during the day than involuntary lard ingestion. If, for instance, the rats selecting lard ate it all during the first 6 h of the dark period but the rats eating the chow-lard mix ate throughout the dark period, then the marked circadian changes in hormone concentrations and autonomic nervous system activity that occur during the daily activity period (21, 22, 23, 24) might have influenced the disposition of the ingested lard calories. Feeding times of rats eating chow only or high-fat diet were similar, and there was little variance across the groups within the times of day that intakes were measured. Although there was considerably more variance in times of feeding in the rats with choice of chow and lard, the timing of the intake resembled the other two groups. Therefore, it does not seem that lard choice markedly affected the daily timing of feeding.
We have previously suggested that mesenteric fat weight is a surrogate for a metabolic-neural feedback signal that damps activity in the HPA axis (9). However, in these studies there were no significant differences in the intra-abdominal or sc fat depot weights, suggesting that the signal is not transmitted by a neural afferent from fat pads (25). Therefore, it seems that the metabolic signal that reduces activity in the HPA axis in times of plenty may not be neural.
In the first, but not second experiment, both ACTH and corticosterone responses to acute restraint were diminished in the rats with prior choice of lard, although when corticosterone responses of rats with choice were compared to those eating the same amount of fat without choice, both ACTH and corticosterone responses were reduced in both experiments. In the second experiment, it seems possible that adrenal sensitivity to ACTH may have been increased in the choice group (26); alternatively, it may be that under conditions of acute restraint ACTH is the more sensitive measure of HPA activity because adrenal responses saturate at fairly low concentrations of ACTH (27).
Total caloric intake was significantly reduced in rats with choice of lard compared with those fed the 50% lard-chow mixture. Both leptin and insulin are signals that act at neurons of the hypothalamic arcuate nuclei to decrease food intake and increase energy loss (28); however, because leptin concentrations were equally increased in both groups eating high fat, this signal does not account for the decrease in total caloric intake in the group with choice. By contrast, insulin concentrations were consistently increased in rats that had the choice of eating lard. Insulin infused into the brain ventricular system inhibits food intake (29). Like leptin, insulin inhibits feeding through inhibition of neuropeptide Y (NPY) expression, a major orexigen, and stimulation of pro-opiomelanocortin expression that results in production of peptides that are strongly anorexigenic (30, 31). It seems plausible that the overall decrease in caloric intake that occurred in the rats with choice of lard, compared with those with no choice is a result of elevated insulin acting on its receptors on arcuate neurons.
Insulin clearly exerts other actions on brain as well as those on the hypothalamus (32, 33, 34, 35, 36, 37). Previously, we have suggested that at low concentrations insulin may increase the preference for lard ingestion based on lard-chow choice studies in rats made diabetic with streptozotocin and then infused with low-dose insulin (5). At similarly low concentrations, it seems as though insulin may also reduce responsivity to acute restraint in the HPA axis. Although changes in hypothalamic peptides regulating feeding can also alter ACTH and corticosterone secretion, both neuropeptide Y and -MSH injected into the brain stimulate activity in the HPA axis (38, 39, 40). Therefore, if insulin is the metabolic signal that reduces HPA responses to acute restraint in these experiments, it may be an action of insulin elsewhere in brain. This possibility remains to be tested directly.
A possible site for an action of insulin in these studies is in the dopaminergic system that appears to signal reward (41, 42). The mesolimbic dopamine-reward system has cell bodies in the ventral tegmental area (VTA) and axons in the nucleus accumbens. Figlewicz et al. (32) have shown that both insulin and leptin receptors are localized on dopaminergic neurons in the VTA, and Figlewicz et al. (43) have also shown that insulin increases expression of dopamine transporters in the VTA. Moreover, axons of the terminal field of this system innervate the prefrontal cortex, a site known to alter responsiveness in the HPA axis (44, 45). Although Figlewicz et al. (46) have made a strong case favoring inhibitory effects of insulin on caloric intake through actions on the mesolimbic reward system, those studies used increases in insulin above normal, through injections of insulin into the ventricular system of rats with normal circulating insulin concentrations. The insulin dose-response curve may be an inverted U, possibly mediated by receptors of differing affinities (47). The two rats in this study that were eliminated from the choice group based on high insulin and ACTH concentrations, markedly overresponded with ACTH to restraint; Figlewicz et al. (46) report inhibition of conditioned place preference for high-fat diet when insulin is directly injected into brain. Together, these results suggest that high insulin concentrations may interfere both with choice-induced inhibition of ACTH as well as conditioned place preference to a high-fat diet. Again, direct tests of this hypothesis are possible and remain to be performed. Finally, palatable food choice clearly activates the opiate systems in brain and nucleus accumbens (48, 49, 50, 51, 52, 53, 54), and it may be through either the dopaminergic or opiatergic systems or, more likely, their interactions, possibly at prefrontal cortex, that the reduced HPA responses are produced (51, 55, 56, 57, 58).
In summary, rats given choice of lard and chow had attenuated ACTH and corticosterone responses to restraint whereas the responses of rats with the same diet but no choice were normal or slightly elevated. Overall caloric intake in the rats with choice was slightly diminished, consistent with the slightly increased insulin concentrations that are known to inhibit caloric intake. However, it seems likely that the inhibition of HPA responses to acute restraint may have been mediated by actions of insulin on the mesolimbic dopamine system that signals pleasure. The output of this system alters activity in the prefrontal cortex that can strongly affect activity in the HPA axis.
Perspective
The fact that choice of lard vs. chow in rats reduces HPA responses to acute restraint whereas, in the absence of choice the same calories from lard do not affect HPA responses may have profound implications for the current epidemic of obesity in humans who live in developed countries. The degree of perceived psychogenic stress is increasing around the world together with increased obesity, type 2 diabetes, cardiovascular morbidity, and mortality (59, 60, 61, 62). Analysis of stress and food choices in a group of students showed that 73% increased ingestion of "snack-type" foods although ingestion of "meal-type" foods (fruits, vegetables, meat, fish) was reported to decrease during stressful periods (63). Because food choice was analyzed in this study, it seems as though stress in people, like rats (6), may decrease overall caloric consumption, but increase the choice of consumption of sweets and savory snacks. As we have suggested previously in our studies of voluntary intake of high sucrose or lard in rats, intake of comfort foods may relieve the sensation of stress as well as the responses to it (6, 9).
Glucocorticoid infusions increase caloric intake in humans (64) as well as in animals (4, 5, 65). After an acute lab psychosocial stressor, women with high cortisol responses to the stressor ate more calories of high density fat and sugar than after a control day (66). Both the incidence of obesity and type 2 diabetes in women are strongly associated with high intake of sugared soft drinks and fruit punches (67), and national consumption of these drinks is increasing (68). However, the obvious down side of this behavior that may be stress-relieving is that with persistent, chronic stressors, obesity, diabetes, and cardiovascular sequelae may occur as a consequence of eating comfort foods. Long-term, prospective studies are needed to determine whether chronic psychosocial stressors change the composition of the foods ingested, if not total calories. It should also be determined, if behavior changes, whether the change results particularly in increased intra-abdominal obesity.
Finally, our finding that lard ingestion has remarkably different effects on HPA axis responses to stress depending on choice, suggests that other systems may be affected as well if rats are given the choice of eating high-density calories. It seems possible that results from studies of rats and other species on forced high-fat diets might differ considerably if the studies were repeated using the choice of fat ingestion.
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Address all correspondence and requests for reprints to: Mary F. Dallman, Department of Physiology, University of California San Francisco, San Francisco, California 94143-0444. E-mail: dallman@itsa.ucsf.edu.
Abstract
Although rats given the choice of eating high-density calories as concentrated sucrose solutions or lard exhibit reduced responsivity in the hypothalamo-pituitary-adrenal axis, rats fed high-fat diets have normal or augmented responses to stressors. To resolve this apparent discrepancy, we compared in adult male rats the effects of 7-d feeding with lard + chow (choice) to feeding a 50% lard-chow mixture (no-choice) and to chow only. Rats with choice composed diets with 50–60% total calories from lard. Rats were exposed to 30 min of restraint on d 7. In the choice group, there was a robust inhibition of ACTH and corticosterone responses to restraint compared with chow or no-choice groups. Total caloric intake was less with choice than no-choice. Fat depot weights and body weight gain were similar in the high-fat groups. Leptin concentrations were equal but insulin was higher in the choice group. We conclude the following: 1) choice of eating high-density calories strongly damps hypothalamo-pituitary-adrenal responses to stress; without choice, high-density diet is ineffective; and 2) insulin may signal metabolic well-being, and may act through hypothalamic sites to reduce caloric intake but through forebrain sites to damp stress responses.
Introduction
IN RECENT YEARS we have provided either separate containers of 30% sucrose or lard, or both, in addition to standard chow, after adrenalectomy (1, 2, 3, 4, 5), and after chronic stressors in intact or adrenalectomized rats (1, 6, 7). In general, the provision of these "comfort" foods reduced activity in the hypothalamo-pituitary-adrenal (HPA) axis, and rats that ate the comfort foods increased mesenteric fat stores. These results led us to propose that an unidentified metabolic feedback signal (8) from central fat stores modulates the HPA response to stress (9).
Although rats that voluntarily compose the percentage of lard in their diets have reduced HPA responses, there is a considerable literature that suggests that a high-fat diet stimulates activity in the HPA axis (10, 11, 12, 13, 14, 15, 16). Moreover, lipid acutely infused into rats also stimulates the HPA axis (17, 18, 19). Experimental resolution of these differences in the reported effects of high-fat feeding on function in the HPA axis is important.
The most obvious difference between our experiments showing inhibition, and those of others showing stimulation of HPA activity is choice or no choice of the rats to eat fat. If such differences can be shown within an experiment, the differences between fat-fed groups might reveal a signal that acts to inhibit activity in the HPA axis. Moreover, in an unpublished experiment (K. D. Laugero and F. Gomez, unpublished data), when we removed choice of drinking sucrose by adding 30% sucrose to the saline solution of adrenalectomized rats, this stimulated, and did not inhibit ACTH as voluntary sucrose ingestion does. Because rats develop a salt appetite after adrenalectomy (20), they drank large amounts of the saline-sucrose mix to obtain salt. Thus, the component of choice seemed as though it might be quite important in the differences observed after various regimens involved in feeding rats a high caloric nutrient.
The current experiments were performed to distinguish between the effects of voluntary and involuntary lard intake for 7 d before intact rats were stressed with an acute 30-min period of restraint. Caloric intake, timing of intake, metabolic hormone concentrations, and fat depot weights were measured as well as the acute ACTH and corticosterone responses to restraint.
Materials and Methods
Animals
Male Sprague Dawley rats, 60 d old, weighing 280–320 g from Bantin and Kingman Suppliers (Freemont, CA) (experiment 1a and 1b) and Simonsen Laboratories, Inc. (Gilroy, CA) (experiment 2) were housed in University of California San Francisco (UCSF) Lab Animal Resources Center in temperature- (21–23 C) and light- (12 h light; lights on at 0700 h) controlled rooms. Rats were housed individually in hanging wire cages and given food (Purina Chow no. 5008; Purina, St. Louis, MO) and water ad libitum until the start of the experiment, at least 4 d after arrival at UCSF. All procedures were approved by the UCSF Institutional Animal Care and Use Committee.
Experimental design
On d 0 at the end of the light cycle (at 1800 h) until d 7, each group was provided with one diet; the experiments differed in the specific array of diets offered. The diets were: a cup of lard (Armour, 9 cal/g) in addition to Purina rodent chow (3.31 cal/g) (choice); a similar cup but with pelleted chow (chow); a moderate-fat diet containing 20% calories from lard and 80% calories from Purina chow (no-choice20%, NC20%; calculated energy content of 4.898 cal/g); or a high-fat diet containing 50% calories from lard and 50% calories from Purina Chow (no-choice50%, NC50%; calculated energy content of 6.155 cal/g). Body weight and food intake was recorded daily in the morning. On d 7 at 0900 h, each rat was removed from his home cage and within 2 min a blood sample was collected from a cut (1 mm) made across the tip of the tail. Rats were placed in restraint tubes for 30 min, were then removed from the tubes and replaced in their home cages. Blood was collected 15, 30, 60, 90, and 120 min after the initial tail nick (0 min). Blood (300 μl) was collected into heparinized Natelson blood collection tubes and kept at 4 C until centrifuged in the cold. Aliquots of plasma were stored at –20 C until assays. All rats were killed 4 h after start of the restraint stress (at 1300 h) by decapitation within 10 sec after they had been taken from their home cages. Trunk blood was collected in tubes with EDTA, kept on ice, and centrifuged in the cold. Aliquots of plasma were stored at –20 C until assayed. Individual mesenteric, epididymal, sc (inguinal), and perirenal white adipose tissues, thymus, and adrenals were dissected, cleaned, and weighed.
Experiment 1a: choice (n = 7), chow (n = 7); experiment 1b: choice (n = 7), chow (n = 6), no-choice20% (n = 7), no-choice50% (n = 7); experiment 2: choice (n = 5), chow (n = 5), no-choice50% (n = 8). The 50% lard- and 20% lard-diets were prepared fresh daily by pulverizing chow pellets, adding the calculated caloric proportion of lard and thoroughly mixing.
Plasma assays
Plasma glucose and triglyceride concentrations were measured in plate assays (5). All plasma hormone concentrations were determined by radioimmunoassay (5).
Statistical analyses
Data were first analyzed by two-way ANOVA. A significant (P 0.05) global effect of ANOVA was followed by one-way ANOVA and post hoc tests of individual group differences [Fisher’s projected least significant difference (PLSD)]. Corticosterone (B) and ACTH data were analyzed by ANOVA corrected for repeated measures over time. Rats were excluded from analysis in experiments 1a (n = 7), 1b (n = 5), and 2 (n = 3) because of missing blood samples (n = 10), food hopper problems (n = 1) or growth failure (n = 2). In addition, two choice rats were excluded because they had very high insulin concentrations (>6 ng/ml) and high basal ACTH as well as ACTH responses to restraint. The final numbers in each group are indicated in the figures.
Results
Experiment 1
Choice groups ate 50% of total calories from lard. To control for this amount of dietary lard, we provided one group of rats in experiment 1b with a mixture of 50% calories from lard with 50% calories from chow to establish a pair-fed group (no-choice50%). A second group was given a lard-chow diet with fewer calories (20%) from lard (no-choice20%) similar to that in a previous study of no-choice (10).
Both the chow and the choice groups in experiments 1a and 1b had similar caloric intake, body weight gain, and hormonal responses to the 30-min restraint. Therefore, we combined the results (Fig. 1).
FIG. 1. Rats with choice of lard intake eat fewer total calories but ingest 50% of those calories as lard. A, Caloric intake during the experiment (groups were provided chow (n = 9), 20% lard-chow mixture (no-choice20%, NC20%; n = 7), choice of chow + lard (n = 9), and 50% lard-chow mixture (no-choice50%, NC50%; n = 6); differences among groups are indicated by different letters). B, Composition of intake of lard as a percentage of the total in the choice group.
Groups on choice, as well as those on the no-choice20% and no-choice50% were hyperphagic compared with the chow group (Fig. 1A). The no-choice20% and no-choice50% groups had greater caloric intake than the choice group. Voluntary lard intake (expressed as percentage of total intake) was stable during the course of the experiment (Fig. 1B). Body weight gain over the 7-d period did not differ among groups (Table 1; ANOVA: F (3, 26) = 0.48; P = 0.7); however, relative fat stores and plasma leptin concentrations were significantly increased in the choice and no-choice50% groups, but not in the no-choice20% group (Fig. 2, A and B). Plasma insulin concentrations were increased in the choice compared with those in the chow, no-choice20% and no-choice50% groups (Fig. 2C). Plasma glucose and triglyceride concentrations were not different among the groups, although plasma free fatty acid concentrations tended to be modestly elevated in the choice and no-choice50% groups compared with the chow group (Table 1; P < 0.1).
TABLE 1. Experiment 1, a and b
FIG. 2. Choice increases fat depots, leptin, and insulin. A, Mesenteric and sc fat depot weights. B, Plasma leptin concentrations. C, Plasma insulin concentrations in groups eating chow (n = 9), medium (20% lard) diet (no-choice20%, NC20%; n = 7), choice (50% lard, 50% chow; n = 9), and high (no-choice50%, NC50%; n = 6) diet. Differences among groups are indicated by different letters above the bars (post hoc PLSD). Note the elevated insulin in the choice group.
Basal plasma ACTH concentrations were similar in all groups (Fig. 3A and Table 1) and exposure to restraint also increased plasma ACTH concentrations significantly. However, the ACTH response to stress was significantly decreased in choice compared with no-choice50% or no-choice20% groups (Fig. 3A). Initial corticosterone concentrations were slightly increased in choice and no-choice50% groups compared with those on chow (Fig. 3B and Table 1). Acute restraint increased plasma corticosterone concentrations significantly in all groups of rats. However, again, the corticosterone response to stress was significantly decreased in the choice compared with chow, no-choice20% and no-choice50% groups.
FIG. 3. Choice attenuates ACTH and corticosterone responses to restraint. A, ACTH responses to restraint (inset, area under the response curve). B, Corticosterone responses to restraint (inset, area under the response curve). Differences among groups are indicated by different letters above the bars (post hoc PLSD); number per group indicated on legend.
Experiment 2
To analyze further the differences between choice and no-choice50% groups, we repeated the experiment and included more food intake measurements to test whether rats with choice consumed their lard at different times of day from the no-choice50% group.
Rats with choice consumed 64 ± 3% of their calories from lard and were hyperphagic compared with the chow group. However, rats with no-choice50% had higher caloric intake compared with the choice group (Fig. 4A). Chow and no-choice50% groups consumed similar percentages of their total intake of calories during the light period, the first half and the second half of the dark period (Fig. 4, B and C). The choice group was similar in timing of intake to the other groups; however, the variance was larger (Fig. 4, B and C).
FIG. 4. Choice decreases overall caloric consumption but does not affect time of day that intake occurs. A, Caloric intake over the 7-d experiment is increased with lard, but less with choice than no-choice. B and C, The times of day in which calories are ingested (as a percentage of total intake) does not depend upon choice of lard intake. Because there was higher variance in the choice than nonchoice groups on caloric intake in B and C, the individual rat numbers are indicated in the choice group for each time of day. Chow, n = 4; choice of chow and lard, n = 6; no choice of chow and lard, n = 5.
ACTH and corticosterone responses to 30-min restraint are shown in Fig. 5, A and B, and the results confirmed the first experiment showing diminished ACTH responses to restraint in the choice group (Fig. 5, A and B). However, the corticosterone response in the choice group was significantly decreased only in the no-choice50% group, and was not different from the chow group in this experiment.
FIG. 5. Choice attenuates ACTH and corticosterone responses to restraint. A, ACTH responses to restraint (inset, area under the response curve). B, Corticosterone responses to restraint (inset, area under the response curve). Differences among groups are indicated by different letters above the bars in the inset (post hoc PLSD); number per group indicated on legend.
Body weight gain did not differ among the groups (Table 2). Relative fat stores and plasma leptin concentrations were increased in both choice and no-choice50% groups (Table 2). Plasma insulin concentrations were increased in the choice compared with those in the chow and no-choice50% groups (Table 2) and plasma adiponectin concentrations increased in the choice and increased slightly with no-choice50% diet (Table 2).
TABLE 2. Results from experiment 2
Discussion
Rats that ate a 50% lard-chow diet had normal HPA responses to acute restraint, whereas those selecting equal proportions of lard and chow had robustly diminished ACTH, and to a lesser extent, corticosterone responses. In the groups of rats voluntarily or involuntarily eating the high-fat diets, sc and intra-abdominal fat depot weights increased, to an equal extent compared with rats eating only chow. Additionally, there was no differential increase in body weight between the groups eating 50% lard mixed with chow or self-selecting the same percentages of lard and chow. Rats eating the lard-chow mixture had consistently increased caloric intake over the 7 d of study compared with the rats with choice. Obtained within experiments, these results confirm the apparently conflicting reports that high-fat diets stimulate HPA responses and that choice of high density calories diminishes HPA responses (see introduction).
We considered the possibility that voluntary lard ingestion might occur at different times during the day than involuntary lard ingestion. If, for instance, the rats selecting lard ate it all during the first 6 h of the dark period but the rats eating the chow-lard mix ate throughout the dark period, then the marked circadian changes in hormone concentrations and autonomic nervous system activity that occur during the daily activity period (21, 22, 23, 24) might have influenced the disposition of the ingested lard calories. Feeding times of rats eating chow only or high-fat diet were similar, and there was little variance across the groups within the times of day that intakes were measured. Although there was considerably more variance in times of feeding in the rats with choice of chow and lard, the timing of the intake resembled the other two groups. Therefore, it does not seem that lard choice markedly affected the daily timing of feeding.
We have previously suggested that mesenteric fat weight is a surrogate for a metabolic-neural feedback signal that damps activity in the HPA axis (9). However, in these studies there were no significant differences in the intra-abdominal or sc fat depot weights, suggesting that the signal is not transmitted by a neural afferent from fat pads (25). Therefore, it seems that the metabolic signal that reduces activity in the HPA axis in times of plenty may not be neural.
In the first, but not second experiment, both ACTH and corticosterone responses to acute restraint were diminished in the rats with prior choice of lard, although when corticosterone responses of rats with choice were compared to those eating the same amount of fat without choice, both ACTH and corticosterone responses were reduced in both experiments. In the second experiment, it seems possible that adrenal sensitivity to ACTH may have been increased in the choice group (26); alternatively, it may be that under conditions of acute restraint ACTH is the more sensitive measure of HPA activity because adrenal responses saturate at fairly low concentrations of ACTH (27).
Total caloric intake was significantly reduced in rats with choice of lard compared with those fed the 50% lard-chow mixture. Both leptin and insulin are signals that act at neurons of the hypothalamic arcuate nuclei to decrease food intake and increase energy loss (28); however, because leptin concentrations were equally increased in both groups eating high fat, this signal does not account for the decrease in total caloric intake in the group with choice. By contrast, insulin concentrations were consistently increased in rats that had the choice of eating lard. Insulin infused into the brain ventricular system inhibits food intake (29). Like leptin, insulin inhibits feeding through inhibition of neuropeptide Y (NPY) expression, a major orexigen, and stimulation of pro-opiomelanocortin expression that results in production of peptides that are strongly anorexigenic (30, 31). It seems plausible that the overall decrease in caloric intake that occurred in the rats with choice of lard, compared with those with no choice is a result of elevated insulin acting on its receptors on arcuate neurons.
Insulin clearly exerts other actions on brain as well as those on the hypothalamus (32, 33, 34, 35, 36, 37). Previously, we have suggested that at low concentrations insulin may increase the preference for lard ingestion based on lard-chow choice studies in rats made diabetic with streptozotocin and then infused with low-dose insulin (5). At similarly low concentrations, it seems as though insulin may also reduce responsivity to acute restraint in the HPA axis. Although changes in hypothalamic peptides regulating feeding can also alter ACTH and corticosterone secretion, both neuropeptide Y and -MSH injected into the brain stimulate activity in the HPA axis (38, 39, 40). Therefore, if insulin is the metabolic signal that reduces HPA responses to acute restraint in these experiments, it may be an action of insulin elsewhere in brain. This possibility remains to be tested directly.
A possible site for an action of insulin in these studies is in the dopaminergic system that appears to signal reward (41, 42). The mesolimbic dopamine-reward system has cell bodies in the ventral tegmental area (VTA) and axons in the nucleus accumbens. Figlewicz et al. (32) have shown that both insulin and leptin receptors are localized on dopaminergic neurons in the VTA, and Figlewicz et al. (43) have also shown that insulin increases expression of dopamine transporters in the VTA. Moreover, axons of the terminal field of this system innervate the prefrontal cortex, a site known to alter responsiveness in the HPA axis (44, 45). Although Figlewicz et al. (46) have made a strong case favoring inhibitory effects of insulin on caloric intake through actions on the mesolimbic reward system, those studies used increases in insulin above normal, through injections of insulin into the ventricular system of rats with normal circulating insulin concentrations. The insulin dose-response curve may be an inverted U, possibly mediated by receptors of differing affinities (47). The two rats in this study that were eliminated from the choice group based on high insulin and ACTH concentrations, markedly overresponded with ACTH to restraint; Figlewicz et al. (46) report inhibition of conditioned place preference for high-fat diet when insulin is directly injected into brain. Together, these results suggest that high insulin concentrations may interfere both with choice-induced inhibition of ACTH as well as conditioned place preference to a high-fat diet. Again, direct tests of this hypothesis are possible and remain to be performed. Finally, palatable food choice clearly activates the opiate systems in brain and nucleus accumbens (48, 49, 50, 51, 52, 53, 54), and it may be through either the dopaminergic or opiatergic systems or, more likely, their interactions, possibly at prefrontal cortex, that the reduced HPA responses are produced (51, 55, 56, 57, 58).
In summary, rats given choice of lard and chow had attenuated ACTH and corticosterone responses to restraint whereas the responses of rats with the same diet but no choice were normal or slightly elevated. Overall caloric intake in the rats with choice was slightly diminished, consistent with the slightly increased insulin concentrations that are known to inhibit caloric intake. However, it seems likely that the inhibition of HPA responses to acute restraint may have been mediated by actions of insulin on the mesolimbic dopamine system that signals pleasure. The output of this system alters activity in the prefrontal cortex that can strongly affect activity in the HPA axis.
Perspective
The fact that choice of lard vs. chow in rats reduces HPA responses to acute restraint whereas, in the absence of choice the same calories from lard do not affect HPA responses may have profound implications for the current epidemic of obesity in humans who live in developed countries. The degree of perceived psychogenic stress is increasing around the world together with increased obesity, type 2 diabetes, cardiovascular morbidity, and mortality (59, 60, 61, 62). Analysis of stress and food choices in a group of students showed that 73% increased ingestion of "snack-type" foods although ingestion of "meal-type" foods (fruits, vegetables, meat, fish) was reported to decrease during stressful periods (63). Because food choice was analyzed in this study, it seems as though stress in people, like rats (6), may decrease overall caloric consumption, but increase the choice of consumption of sweets and savory snacks. As we have suggested previously in our studies of voluntary intake of high sucrose or lard in rats, intake of comfort foods may relieve the sensation of stress as well as the responses to it (6, 9).
Glucocorticoid infusions increase caloric intake in humans (64) as well as in animals (4, 5, 65). After an acute lab psychosocial stressor, women with high cortisol responses to the stressor ate more calories of high density fat and sugar than after a control day (66). Both the incidence of obesity and type 2 diabetes in women are strongly associated with high intake of sugared soft drinks and fruit punches (67), and national consumption of these drinks is increasing (68). However, the obvious down side of this behavior that may be stress-relieving is that with persistent, chronic stressors, obesity, diabetes, and cardiovascular sequelae may occur as a consequence of eating comfort foods. Long-term, prospective studies are needed to determine whether chronic psychosocial stressors change the composition of the foods ingested, if not total calories. It should also be determined, if behavior changes, whether the change results particularly in increased intra-abdominal obesity.
Finally, our finding that lard ingestion has remarkably different effects on HPA axis responses to stress depending on choice, suggests that other systems may be affected as well if rats are given the choice of eating high-density calories. It seems possible that results from studies of rats and other species on forced high-fat diets might differ considerably if the studies were repeated using the choice of fat ingestion.
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