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Endocrinology Vol. 144, No. 7 2941-2946
Copyright © 2003 by The Endocrine Society

Increased Dietary Fat Attenuates the Anorexic Effects of Intracerebroventricular Injections of MTII

Deborah J. Clegg, Stephen C. Benoit, Ellen L. Air, Alana Jackman, Patrick Tso, David D’Alessio, Stephen C. Woods and Randy J. Seeley

Departments of Psychiatry (D.J.C., S.C.B., E.L.A., S.C.W., R.J.S.), Biomedical Sciences and Cell Biology (E.L.A.), Pathology (P.T.), and Medicine (D.D’A.), University of Cincinnati Medical Center, Cincinnati, Ohio 45267-0559; and Procter & Gamble Pharmaceuticals (A.J.), Cincinnati, Ohio 45040

Address all correspondence and requests for reprints to: Deborah J. Clegg, Ph.D., Department of Psychiatry, University of Cincinnati Medical Center, P.O. Box 670559, Cincinnati, Ohio 45267-0559. E-mail: debbie.clegg{at}uc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The hypothalamic melanocortin (MC) system provides a critical inhibitory control on food intake and body weight. Because access to high-fat (HF) diets is associated with the development of obesity, we hypothesized that increased dietary fat attenuates signaling through the MC system. To evaluate this hypothesis, we compared the efficacy of the MC3/4 receptor agonist, MTII, to reduce food intake in rats fed carefully matched HF or low-fat (LF) diets for 12 wk. Rats given the HF diet ad libitum were significantly more obese than rats given the LF diet, and had significantly higher plasma insulin and leptin levels. MTII given into the third cerebral ventricle in doses of 0.1, 0.3, and 1.0 nmol was less effective at reducing food intake in HF rats than in LF rats. Whole-hypothalamic expression of the MC agonist precursor gene, proopiomelanocortin, the MC antagonist agouti-related protein, and the MC4 receptor, were not different between the HF and LF groups. These results indicate that consumption of a HF diet decreases signaling through the melanocortin system, an abnormality that could contribute to diet-induced obesity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OBESITY IS A MAJOR health problem (1), and as the incidence continues to rise, the obesity-related costs are also increasing (2, 3, 4, 5, 6, 7). Although genetic factors contribute to the propensity of an individual to become obese, environmental factors are also important (4, 8, 9). Several lines of evidence point to consumption of high-fat (HF) diets as an important environmental factor predisposing to obesity. Epidemiological studies have identified a significant positive correlation between average dietary fat intake and the incidence of obesity (10, 11, 12, 13, 14, 15, 16, 17, 18). Whereas body weight is tightly regulated, experimental studies in rodents and humans suggest that a diet that is high in fat alters the negative feedback system that normally regulates body fat, resulting in increased adipose stores (see Refs. 14, 18, 19, 20, 21, 22, 23, 24, 25). These data collectively suggest that chronic intake of different amounts of fat affects body weight by interacting with the fundamental processes determining energy balance.

A critical unanswered question is how consumption of a HF diet modifies the negative feedback system that controls body fat. In principle, chronic exposure to a HF diet could impact a number of systems that contribute to normal energy homeostasis. We have previously described a paradigm that begins to address the role of fat in the regulation of food intake and body weight by feeding rats experimental diets that differ only in the percentage of calories that are derived from fat and carbohydrate, with protein and other nutrients carefully matched on per kilocalorie basis for both diets (25). The HF diet contains 40% of calories from fat, primarily butter oil (1/20th of the fat comes from soybean oil to ensure that all essential fatty acids are in the diet) and 45% of calories from carbohydrate. The low-fat (LF) diet has only 8% of calories from fat, but a greater percentage of energy from sucrose and other carbohydrates (75% of total calories come from carbohydrates). Rats maintained on the HF diet have a 10% increase in body weight, and a 50% increase in total body adiposity over 10 wk. Moreover, HF rats are hyperleptinemic, hyperinsulinemic, and insulin resistant, similar to what is observed in human obesity (25).

Evidence from studies in rodents and humans has directly implicated the hypothalamic melanocortin (MC) peptide system as a critical control system for maintenance of normal energy homeostasis (26, 27, 28, 29, 30, 31). Separate populations of neurons within the arcuate nucleus of the hypothalamus express the precursor of MC receptor agonists, proopiomelanocortin (POMC), and MC receptor antagonists, agouti-related protein (AgRP) (32, 33, 34). The biological effects of MC peptides are diverse and largely mediated through one of five MC receptor isoforms, of which MC3 receptor (MC3R) and MC4 receptor (MC4R) have a functional role in appetite and body fat regulation.

The MC4R is widely expressed in the brain in regions of the hypothalamus that are known to control feeding behavior, such as the paraventricular nucleus, dorsal medial hypothalamus, and the lateral hypothalamus (35, 36, 37, 38). The MC4R has a pivotal role in controlling feeding behavior. Targeted disruption of the MC4R results in hyperphagia and obesity in rodents and humans (39, 40, 41). Additionally, it has been estimated that up to 5% of morbidly obese humans have spontaneous mutations in the MC4R gene (42). MC3R-deficient mice display an increase in adipose mass, but they are neither overweight nor hyperphagic (43, 44), further supporting a role for the MC4R in food intake and energy balance. An endogenous agonist of MC3R and MC4R is {alpha}-MSH (30, 45, 46), one of the posttranslationally cleaved bioactive peptides from POMC (reviewed in Ref. 29). Other bioactive peptides of POMC include ACTH, and ß-endorphin (reviewed in Ref. 29). Central administration of {alpha}-MSH robustly decreases food intake in rats (47, 48). MTII is a synthetic analog of {alpha}-MSH that also has agonistic properties at both MC3R and MC4R. Central administration of MTII also results in decreased food intake, and leads to reductions in body weight in mice and rats (26, 49). However, administration of MTII to MC4R knockout mice does not reduce food intake or body weight (50), further suggesting a role for the MC4R in food intake and body weight homeostasis.

The aim of the present study was to test the hypothesis that chronic ingestion of a HF diet acts in part to increase body fat by reducing signaling through the central nervous system MC system. To test this hypothesis, we performed a dose-response study with MTII in rats maintained on HF or LF diets. We also analyzed hypothalamic mRNA expression of AgRP, POMC, and MC4R in these rats. The results indicate that chronic exposure to a HF diet attenuates the ability of MTII to suppress food intake, suggesting that the MC pathway is involved in diet-induced obesity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
All procedures were approved by the Institutional Animal Care and Use Committee of the University of Cincinnati and were conducted in AAALAC-approved facilities. Male Long-Evans rats (220–250 g; Harlan, Indianapolis, IN) were housed individually in plastic tub cages and maintained on a 12-h light, 12-h dark schedule at constant temperature (20 C). They received pelleted chow (Purina, St. Louis, MO) and water ad libitum for 1 wk before being assigned to groups to allow for recovery from shipment and stabilization of body weight before being divided into dietary groups (n = 15 per group) on the basis of comparable mean body weight.

Diets
Two pelleted semipurified, nutritionally complete experimental diets were prepared by Dyets, Inc. (Bethlehem, PA). The HF diet contained 20% fat by weight (19% butter oil and 1% soybean oil to provide essential fatty acids), and 45% of total calories from carbohydrates, for a total of 4.4 kcal/g. The LF diet contained 3% butter oil and 1% soybean oil by weight, having 75% of calories from carbohydrates, and 3.6 kcal/g. The two diets had the same amount of protein per calorie of diet as well as equal amounts of all of the essential minerals and vitamins required for rats as indicated in AIN-93 (see Table 1Go); thus, they differed only by fat and carbohydrate content.


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  		Table 1. Macronutrient composition of LF, HF, and regular chow diets by kilocalories per gram and by percent of total kilocalories

 
Experimental groups
The two groups used for comparisons are those that received the HF or LF diets ad libitum (HF and LF groups, respectively).

Cannulations
After 8 wk on the HF or LF diet, cannulas were placed under stereotaxic guidance into the third cerebral ventricle (i3vt) of the rats as described previously (51). Briefly, rats were anesthetized with ketamine/xylazine (10:6.5 solution; 1.0 ml/kg) and placed into a stereotaxic frame. A 22-gauge guide sleeve was lowered along the midsagittal plane into the third ventricle according to the coordinates of Paxinos and Watson (2.2 mm caudal to bregma and 7.5 mm ventral to the sinus, with {lambda} and bregma at the same vertical coordinate) and then secured to the skull with screws and dental acrylic. Following recovery of a minimum of 7 d and return to maintenance body weight, cannula placement was confirmed via injection of 10 ng angiotensin through a cannula extending 1 mm beyond the tip of the guide sleeve (i3vt injection). Only those animals that consumed at least 5 ml water within 30 min of injection were deemed to have correct placement and were used in the subsequent studies.

Water and food consumption
All animals were habituated to daily handling, and administration of i3vt injections of physiological saline (1 µl) at the same time each day (see below). The design was within-subjects, with each rat receiving all doses of MTII plus vehicle in an order that was counterbalanced across subjects. We allowed full recovery to the animal’s previous body weight and a minimum of 7 d before the next injection series.

Procedure
Experimental drugs were physiological saline, or MTII in saline (0.1, 0.3, 1.0 nmol/rat, Phoenix Pharmaceuticals, Inc., Mountain View, CA), each in 1 µl. Rats received the injections 1 h before lights out. For these assessments, food but not water was removed for one hour before the injections and returned immediately after the injections. Food and water intake were assessed 2, 4, and 24 h after injection.

At the time of cannulation, a separate cohort of animals, 10 animals per diet group, was food deprived 4 h before the onset of the dark to establish consistent baselines for insulin and glucose measurements. One hour before the onset of dark, the rats were killed by CO2 anesthesia and decapitation. Trunk blood was collected in EDTA-coated tubes. Plasma was isolated from whole blood by centrifugation and stored at –20 C until analysis. Hypothalamic tissue was extracted from whole brain and was immediately frozen on dry ice and stored at -80 C until RNA extraction.

Hypothalamic RNA extraction
Brains were quickly removed and the hypothalamus dissected free, then immersed in liquid nitrogen at –80 C until used. Each hypothalamus was homogenized in 1 ml TriReagent (Molecular Research Center, Inc., Cincinnati, OH) with a glass-col tube and Teflon pestle for 1 min at 4000 rpm, followed by RNA extraction according to manufacturer’s instructions. RNA was resuspended in RNA-Secure (Ambion, Inc., Austin, TX) and deoxyribonuclease treated with DNA-Free (Ambion, Inc., Austin, TX).

Taqman RT-PCR
RNA samples were plated at 10 ng in triplicate on a 96-well Optical Reaction plate (Perkin-Elmer Applied Biosystems, Foster City, CA). One step RT-PCR was performed on the Perkin-Elmer Prism 7700 Sequence Detector System for 40 cycles. The EZ RT-PCR core reagent kit (Perkin-Elmer Applied Biosystems) was used to generate reaction mastermix for multiplex reactions, where expression data were normalized to the reference gene glyceraldehyde-3-phosphate dehydrogenase. The following optimized primers and probes were used. For POMC, the forward primer was 5'-CGC CCG TGT TTC CA-3', and the reverse primer 5'-TGA CCC ATG ACG TAC TTC C-3' and the 6 FAM probe 5'-CG GAG ATG AAC AGC CCT TGA CT TAMRA-3'. For glyceraldehyde-3-phosphate dehydrogenase, the forward primer was 5'-TGC ACC ACC AAC TGC TTA G-3', and the reverse primer was 5'-GGA TGC AGG GAT GAT GTT C-3' and the VIC probe 5'-VIC CAG AAG ACT GTG GAT GGC CCC TC TAMRA-3'. For AGRP, the forward primer was 5'-TTC CCA GAG TTC TCA GGT CTA-3', and the reverse primer was 5'-ATC TAG CAC CTC TGC CAA A-3' and the FAM probe was 5'-FAM CTG AAG AAG ACA GCA GCA GAC CG TAMRA-3'. For MC4-R, the forward primer was 5'-AGA ATT TGT CAC TCA GGC AC-3', and the reverse primer was 5'-TAT TTT CCA ACC AAA GCA CTA T-3' and the FAM probe was 5'-FAM ACC TGAGCA GTG TAC TTC CCA ACA G TAMRA-3' (Perkin-Elmer Applied Biosystems). AgRP and POMC plates were run using 10 ng of RNA on for 40 cycles, whereas the MC4-R was run using 250 ng RNA on 45 cycles.

The results were transferred and analyzed in Excel using the delta delta CT method (Perkin-Elmer Applied Biosystems).

Plasma analyses
The immunoreactive insulin assay used a guinea pig antiinsulin serum with high affinity for rodent insulin (52). Plasma leptin was measured using a rat leptin RIA kit (Linco Research, Inc., St. Louis, MO).

Data analyses
Food intake data were analyzed by parametric statistics (repeated measures ANOVA with time as the repeated measure, followed by planned t tests). The expression of RNA was analyzed by comparing the HF to the LF group using one-way ANOVA and Newman-Keuls Multiple Comparison Test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Body weight and plasma regulatory hormones
Animals maintained on the HF diet were significantly heavier than animals fed the LF diet, confirming our previous findings (Fig. 1Go). Consistent with the effects on body weight, rats fed the HF diet had significantly elevated plasma immunoreactive insulin (Fig. 2AGo, P < 0.05) and immunoreactive leptin (Fig. 2BGo, P < 0.05) relative to the LF group.



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  		Figure 1. HF diet induces weight gain. Body weight for Long Evans rats maintained on either a HF diet (HF, n = 30) or LF-fed animals (LF, n = 30) over 90 d ± SEM. Significant differences in body weight were observed in the HF-fed animals (*, P < 0.05).

 


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  		Figure 2. HF diet elevates plasma insulin and leptin. A, Fasting plasma insulin. Fasting plasma insulin from Long Evans rats maintained on HF (n = 20) or LF (n = 20) diets. Bars are means ± SEM; plasma insulin levels were significantly higher in the HF group (*, P < 0.05). B, Fasting plasma leptin. Fasting plasma leptin from Long Evans rats maintained on HF (n = 20) or LF (n = 20) diets. Bars are means ± SEM; HF-fed rats had significantly higher plasma leptin (* P < 0.05).

 
Food intake in response to MTII
Figure 3AGo depicts the 4-h intake (intake after 2 h is comparable, data not depicted), and Fig. 3BGo depicts 24-h food intakes in response to i3vt MTII (0.1, 0.3, 1.0 nmol/1 µl saline) in the HF and LF groups. All doses of MTII significantly reduced 4-h intake relative to saline in the LF rats (P < 0.05, Fig. 3AGo). In contrast, rats maintained on the HF diet were less sensitive to the effects of 0.1 nmol of MTII. At 24 h, the HF rats demonstrated resistance to the anorectic effect of MTII in that the lowest doses (0.1 and 0.3; Fig. 3BGo) no longer inhibited food intake. Both groups of rats lost significant body weight over the 24 h following the largest dose of i3vt MTII (data not depicted). Food intake over 24 h for HF rats following saline injection was 14.8 ± 1.7; following 0.1 MTII was 10.1 ± 2.2; following 0.3 MTII was 7.6 ± 2.4; and following 1.0 MTII was 5.7 ± 1.3 g. For animals on the LF diet, 24-h food intake following saline was 19.2 ± 2.3; for 0.1 MTII was 9.2 ± 0.96; for 0.3 MTII was 6.5 ± 0.5; and for 1.0 MTII was 7.1 ± 1.7 g. These data suggest that consumption of a HF diet attenuates responsiveness to centrally injected MTII.



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  		Figure 3. HF diet attenuates sensitivity to MTII. Long Evans rats were fed either a HF diet (HF, n = 20) or LF diet (LF, n = 20). Rats were administered MTII (0.1, 0.3, 1.0 nmol/1 µl) or saline alone (1 µl) i3vt on different days the order being random for each rat. A, Food intake following MTII or saline at 4 h. B, Twenty-four-hour food intake. Bars are means ± SEM; significant reductions in food intake is denoted as *, P < 0.05 relative to saline.

 
Analyses of mRNA expression in animals maintained on a HF or LF diet
We determined hypothalamic gene expression from rats killed 1 h before the onset of the dark. There were no significant differences in expression of AgRP (Fig. 4Go), POMC (Fig. 5Go), or MC4R (Fig. 6Go) between the HF and LF rats, despite animals being heavier on the HF diet.



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  		Figure 4. Dietary intake does not alter hypothalamic mRNA expression of AgRP. Hypothalamic mRNA expression for AgRP in Long Evans rats maintained on HF (n = 10) or LF (n = 10) diets. Bars are means ± SEM; no differences in hypothalamic expression were found across treatments. AgRP expression is expressed in arbitrary units relative to the LF diet condition.

 


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  		Figure 5. Dietary intake does not alter hypothalamic mRNA expression of POMC. Hypothalamic mRNA expression for POMC in Long Evans rats maintained on HF (n = 10) or LF (n = 10) diets. Bars are means ± SEM; POMC expression is expressed in arbitrary units relative to the LF diet condition. *, P < 0.05.

 


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  		Figure 6. Dietary intake does not alter hypothalamic mRNA expression of MC4R. Hypothalamic mRNA expression for MC4R in Long Evans rats maintained on HF (n = 10) or LF (n = 10) diets. Bars are means ± SEM; no differences in hypothalamic expression were found across treatments. MC4R expression is expressed in arbitrary units relative to the LF diet condition.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
As we have previously reported, rats exposed to this HF diet increase their caloric intake and gain weight over a 10-wk regimen (25). Moreover, rats on the HF diet have increased body fat and a concomitant increase in plasma levels of both leptin and insulin. The purpose of these experiments was to test the hypothesis that increased food intake and weight gain on the HF diet is a result of decreased central nervous system MC tone. To that end, we determined the response of HF and LF rats to various doses of the MC receptor agonist, MTII. Rats fed the HF diet had a smaller anorexic response to MTII, suggesting that chronic HF feeding alters signaling through this pathway.

An important question is whether the change in sensitivity to the MTII is a result of exposure to the HF diet or the resulting obesity that changes a number of parameters including plasma insulin and leptin. MTII reliably suppressed food intake for both groups of rats after 4 h. However, the degree of anorexia was dependent on the macronutrient content of the diets. That is, MTII was less effective in reducing food intake in rats maintained on a HF diet. Finally, we assessed the hypothalamic mRNA expression for POMC, MC4R, and AgRP. We found that despite reduced sensitivity to MTII, the HF and LF animals had comparable expression values for POMC, AgRP, and MC4R.

A change in sensitivity to a MC agonist could be caused by several factors. The most obvious of these would be decreased hypothalamic expression of the MC4R. However, this did not occur. An alternative means of decreasing sensitivity of MC signaling would be increased expression of the endogenous MC receptor antagonist, AgRP. But this mechanism also cannot be invoked to explain our findings because AgRP gene expression was not altered. Future experiments will need to address the hypothesis that HF diets reduce MC sensitivity downstream of the MC4R.

The alteration in sensitivity to the actions of MC agonists is not the only way in which changes in the MC system could contribute to the obesity caused by exposure to the HF diet. Reduced MC signaling could be the result of decreased available MC agonist. To test this hypothesis, we measured the expression of the MC agonist precursor, POMC. We found that POMC gene expression did not differ between the HF and LF groups. However, POMC gene expression is normally increased by both leptin and insulin (53, 54, 55, 56, 57). Thus, we would predict that POMC gene expression should have been increased in the HF group. Because of this, it may be that POMC gene expression is inappropriately low given the ambient insulin and leptin levels. However, our results are consistent with previous reports that indicate that neither fat/carbohydrate balance nor dietary fatty acid profile effects POMC expression (58, 59, 60).

Alternatively, it is possible that exposure to a HF diet causes a reduction in POMC expression and that the decreased MC signaling consequently results in increased food intake. The increased food intake continues until increases of body fat stimulate enough leptin to increase MC signaling back to the level of that in LF-fed rats. One interpretation of these findings is that HF feeding leads to a relatively ineffective stimulation of POMC by circulating hormones that regulate energy balance.

Differences in endogenous MC function may also be mediated at a posttranslational, peptide level. Specifically POMC is cleaved into different products including ß-endorphins and {alpha}-MSH. {alpha}-MSH mediates a net catabolic response, whereas ß-endorphin signals the positive hedonic value of food and elicits a net anabolic response (61). Our diets differ in macronutrient content (specifically, in the relative amounts of fat and carbohydrate), and they may also differ with respect to palatability or hedonic components. Thus, rats on the HF diet may be less sensitive to exogenously administered MTII because of increased {alpha}-MSH present at the MC4R. Alternatively, the differences may be related to the palatability of the diet and therefore associated with increased levels of ß-endorphins. Consistent with this, Torri et al. (60) reported that animals allowed access to a cafeteria diet made up of highly palatable foods (12% fat) had increased levels of POMC mRNA and became obese. These findings were in contrast to the animals maintained on a HF diet (20% calories from fat) that were also obese but did not have increased POMC gene expression. The major difference between their two diets was the higher palatability and variety of the cafeteria diet, which may, therefore, have had a higher positive hedonic impact compared with the other HF diet. Their conclusion was that the observed increase in POMC mRNA levels in the cafeteria-fed animals was from a potentiation of ß-endorphin gene expression resulting from the hedonic impact of palatable food in the cafeteria diet (60). These findings and ours begin to address important questions about whether weight gain/obesity per se is responsible for changes in hypothalamic gene expression, or if there are changes directly related to the type of diet consumed. In the absence of studies on POMC-derived peptide release, it is not possible to distinguish directly between opioid or MC effects derived from changes in POMC mRNA.

In these studies, we determined that rats maintained on a HF diet were less sensitive to i3vt MTII than rats fed a LF diet. Our study agrees with the recent report by Pierroz et al. (62) that in mice maintained on a HF diet that became obese, MTII was still effective in reducing food intake and body weight. However, they did not compare the relative effectiveness of MTII to reduce food intake and body weight in animals on a LF or a chow diet. Our study indicates that there is a rightward shift in the dose-response curve for animals maintained on a HF diet.

The current results do not support the possibility that large-scale changes in the hypothalamic MC system result from exposure to a HF diet. Rather, we observed a reduction in sensitivity to MTII’s anorexigenic effects in animals on a HF diet, without apparent changes in POMC, AgRP, or MC4R expression. Our data therefore imply that MC receptor agonists could be useful to treat increased weight that results from exposure to a diet that mimics the macronutrient content of the average American (10, 11, 12, 13, 14, 15, 16, 17, 18). It is important to note, however, that the response to the HF diet is a subtle but cumulative effect. Although rats on the HF diet accumulate 50% more body fat than LF rats, they do so by consuming just 17% more calories over a 10-wk period (25). Thus, the effect of the HF diet is quite subtle. Although the increase in obesity in our society is startling in its rapidity, it also is the result of cumulative changes that cause individuals to be in chronic mild positive energy balance year after year. Therefore, we may need to acknowledge that the search for what underlies the ability of specific diets to increase body weight may be the search for subtle effects such as those observed here.


   Footnotes
 
This work was supported by NIH Grants DK-17844, DK-56863, and DK-54080. The Obesity Research Center at the University of Cincinnati is supported in part by the Procter & Gamble Co.

Abbreviations: AgRP, Agouti-related protein; HF, high-fat; i3vt, third cerebral ventricle; LF, low-fat; MC, melanocortin; MC3R, MC3 receptor; MC4R, MC4 receptor; POMC, proopiomelanocortin.

Received December 31, 2002.

Accepted for publication April 2, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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