Effects of dietary fat types on body fatness, leptin, and ARC leptin receptor, NPY, and AgRP mRNA expression

Hongqin Wang, Len H. Storlien, and Xu-Feng Huang

Metabolic Research Center, Department of Biomedical Science, University of Wollongong, Wollongong, New South Wales 2522, Australia


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Some, but not all, fats are obesogenic. The aim of the present studies was to investigate the effects of changing type and amount of dietary fats on energy balance, fat deposition, leptin, and leptin-related neural peptides: leptin receptor, neuropeptide Y (NPY), agouti-related peptide (AgRP), and proopiomelanocortin (POMC), in C57Bl/6J mice. One week of feeding with a highly saturated fat diet resulted in ~50 and 20% reduction in hypothalamic arcuate NPY and AgRP mRNA levels, respectively, compared with a low-fat or an n-3 or n-6 polyunsaturated high-fat (PUFA) diet without change in energy intake, fat mass, plasma leptin levels, and leptin receptor or POMC mRNA. Similar neuropeptide results were seen at 7 wk, but by then epididymal fat mass and plasma leptin levels were significantly elevated in the saturated fat group compared with low-fat controls. In contrast, fat and leptin levels were reduced in the n-3 PUFA group compared with all other groups. At 7 wk, changing the saturated fat group to n-3 PUFA for 4 wk completely reversed the hyperleptinemia and increased adiposity and neuropeptide changes induced by saturated fat. Changing to a low-fat diet was much less effective. In summary, a highly saturated fat diet induces obesity without hyperphagia. A regulatory reduction in NPY and AgRP mRNA levels is unable to effectively counteract this obesogenic drive. Equally high fat diets emphasizing PUFAs may even protect against obesity.

saturated fat; n-3 polyunsaturated fat; n-6 polyunsaturated fat; hypothalamus; leptin


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

NEUROPEPTIDE Y (NPY) is a potent, centrally acting orexigenic peptide with a high concentration in the hypothalamic arcuate nucleus (Arc). There is considerable evidence of a role for NPY in regulation of food intake and energy balance. This has recently been reviewed (1).

A number of studies (5, 6, 9, 17), although not all (13), have demonstrated that a high-fat diet can induce decreased Arc NPY mRNA expression in rodents. The level of NPY in Arc is regulated by leptin (4). Leptin is a signal protein produced by adipocytes and is positively correlated to the size of body mass (27). Intracerebroventricular injection of leptin significantly reduces the level of Arc NPY mRNA expression in mouse Arc (26). Conversely, overexpression of Arc NPY mRNA has been reported in mice that lack leptin (ob/ob) and in leptin receptor-deficient db/db mice (14, 18). Therefore, it is possible that any reduction in NPY message level induced by high-fat feeding is due to either a direct effect or an indirect effect via increased circulating leptin levels subsequent to the fat-induced positive energy balance and white adipose accumulation.

In a further complexity, it has been reported that the majority of Arc NPY neurons produce agouti-related peptide (AgRP) (7). Functionally, AgRP has a similar effect to that of NPY in promoting energy intake and decreasing energy expenditure, albeit via a different mechanism. AgRP is an antagonist of alpha -melanocortin-stimulating hormone (alpha -MSH) acting at the level of melanocyte receptor subunit 4 (MCR4) (11). In mice, lack of functional leptin (ob/ob) produces a fivefold increase in Arc AgRP mRNA expression, which can be reversed by leptin treatment (20). These studies show that leptin plays an inhibitory role in controlling the production of AgRP. Nutritional status is also important in the regulation of AgRP production, as it has been demonstrated that food deprivation can dramatically increase AgRP mRNA expression in mice (16).

The aim of this study is to investigate the effects of altering both the level and type of dietary fat on the level of leptin and leptin-related hypothalamic neuropeptide mRNA expression, including NPY, AgRP, alpha -MSH, and the leptin receptor.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Three-week-old C57Bl/6J male mice were obtained from the Animal Resource Centre (Perth, Australia; the mouse genomic background was tested by allozyme electrophoresis and random amplified polymorphic DNA-PCR analysis). Mice were housed individually in a temperature-controlled room (20 ± 2°C) with a 12:12-h light-dark cycle (lights on at 0700) and were given ad libitum access to tap water throughout the study.

Diets and experimental procedure. Mice were fed standard laboratory chow for the 1st wk to allow them to adjust to the new environment. Mice were then randomly assigned to a low-fat (10% of calories as fat) or one of the high-fat diets shown in Fig. 1. In the present study, three groups of mice were fed the same concentration (58% kcal) of fat, but different fat types (Table 1). In mice fed a highly saturated fat diet, edible tallow (52°C melting point; Unilever, Australia) and safflower oil (Meadow Lea Foods Australia) contributed equally to fat calories. In mice fed high n-3 polyunsaturated high-fat (PUFA) diets, fish oil (EPA-28; from Yamanouchi Pharmaceutical, Tokyo, Japan) was the fat source. In mice fed a high n-6 PUFA diet, safflower oil contributed all 58% of calories. The saturated fat diet was thus high in saturated fat, with a high n-6-to-n-3 (n-6/n-3) PUFA ratio. The n-3 PUFA diet was almost equally high in saturated fat, but with a high level of n-3 PUFAs and a very low n-6/n-3 PUFA ratio. The n-6 PUFA diet was the most unsaturated, but with a very high n-6/n-3 PUFA ratio. Detailed compositions of the respective diets are as detailed previously (24). Diets were freshly made every week and stored at 4°C. Mice were given food at 1600 each day. Food consumption was measured daily, and body weight was measured weekly.


View larger version (35K):
[in this window]
[in a new window]
 
Fig. 1.   Dietary groups of mice used in this study. Mice were fed highly saturated (HS), n-6 polyunsaturated (PUFA) (n-6), n-3 PUFA (n-3), and low-fat (LF) diets for 1 and 7 wk; or mice fed a high n-3 PUFA (HS-n3) or low-fat (HS-LF) diet after a 7-wk highly saturated fat diet.


                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Composition of the HS, n-3, n-6, and LF diets

After being fed with a high-fat or a low-fat diet for 1, 7, or 11 wk, mice were killed by an overdose of pentobarbitone sodium anesthesia (120 mg/kg ip) between 0700 and 1000. The total carcass was weighed, and adipose tissue (epididymal, perirenal, and inguinal) was dissected free and also weighed. Blood was taken by heart puncture, and plasma was stored at -20°C for later analysis of leptin levels. The brains were removed quickly, frozen immediately in liquid nitrogen, and stored at -70°C. The brains were cryostat sectioned (12-µm-thick coronal sections) at -15°C and mounted on slides. To gain consistency of tissue sections to react with the solution, brain sections from the mice of different dietary groups were mounted on a single slide. That is, each slide had four sections derived from the mice of saturated, n-3 PUFA, n-6 PUFA, and low-fat diets at a single feeding time point. After fixation in 4% paraformaldehyde in PBS (pH 7.4), sections were dehydrated with ethanol and stored at -70°C until used.

Plasma leptin level analysis. Plasma leptin was measured using an RIA kit for mouse leptin (Linco, St. Louis, MO).

In situ hybridization. The specific antisense oligonucleotide probes (Life-Tech, Victoria, Australia) used were 1) 5'-gag tag tat ctg gcc atg tcc tct gct ggc gcg tcc tcg ccc gg-3' for NPY (nucleotide number 1650-1693 of Gene Bank sequence M15792), 2) 5'-TGC AGC AGA ACT TCT TCT GCT CGG TCT GCA GTT GTC TTC TTG AGG-3' (MMU89486, 411-455) and 5'-TGC TTG CGG CAG TAG CAA AAG GCA TTG AAG AAG CGG CAG TAG CAC-3' for AgRP (MMU89486, 763-806), and 3) 5'-cgt tct tga tga tgg cgt tct tga aga gcg tca cca ggg gcg tct-3' for proopiomelanocortin (POMC; J00612, 547-591), and 5'- GCT AAC TCG GTC ACT CAC AAT GCT GTA CTG TAT CTC AGG GA-3' (MMU 49110, 1121-1161) and 5'-AAT TCA GCA TAG CGG TGA TGG CAC GCC TGC TCA TTG CAG CAG T-3' (MMU42464, 1286-1328) for the leptin receptor. No sequences bearing significant homology to the designed probes were found in the Gene Bank (NCBA). All oligonucleotide probes were terminally labeled using a 10-fold molar excess of [35S]dATP (specific activity 1,000 Ci/mmol; Amersham, Buckinghamshire, UK) and terminal transferase (Promega, Madison, WI) and purified over a MicroSpin G-50 spin column (Amersham). The probe concentration was 107 pcm of 35S-labeled probes in 750 µl of hybridization solution, and specificity was confirmed previously (10).

The hybridization was carried out by incubating the sections in the hybridization buffer (50% deionized formamide, 4× SSC, 10% dextran sulfate, 1× Denhardt's solution, 0.2% sheared salmon sperm DNA, 0.1% long-chain polyadenylic acid, 0.012% heparin, 20 mM sodium phosphate, pH 7.0, 1 mM sodium pyrophosphate, 106/75 µl of labeled probe, and 5% dithiothreitol) at 37°C for 16 h. Nonspecific hybridization was determined by including 100-fold molar excess of nonlabeled probes in the respective hybridization solution. After hybridization, sections were washed in 1× SSC buffer at 55°C three times for 20 min each, followed by 1 h of incubation in 1× SSC buffer at room temperature. Finally, sections were dipped sequentially in Milli-Q water, 70% ethanol, and 95% ethanol before air-drying and exposure to Hyper-beta -max film (Amersham UK). After exposure for 2 wk, X-ray films were developed using standard procedures.

Quantification and data analysis. All films were analyzed by using a computer-assisted image analysis system, Multi-Analysis, connected to a GS-690 Imaging Densitometer (Bio-Rad). Quantification of mRNA expression levels in various brain regions was obtained by measuring the average density of each region in five adjacent brain sections, and then the values were compared against a 14C-labeled autoradiographic standard (Amersham UK). All data are shown as means ± SE for groups based on a minimum of five mice in each group. Analyses by ANOVA, followed where appropriate by Dunnett's test, were performed using the JMP statistical package (SAS Institute, Cary, NC).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Food intake. After 1 and 7 wk of the diets, no significant differences were found in a total cumulative calorie intake in mice fed saturated, n-6 PUFA, and n-3 PUFA diets (Fig. 2, A and B). However, from Fig. 3A, it can be seen that among high-fat groups from weeks 5-7, the saturated fat diet group showed a lower intake than those of the PUFA groups [F(2, 21) = 3.76, P = 0.04]. In contrast, Fig. 3B showed that mice on a saturated fat diet always had a higher body weight gain throughout all time points examined. In addition, the low fat-fed group did show a significantly lower cumulative energy intake compared with all high-fat diet groups after 7 wk of diet. Four weeks of dietary reversal show that a change in the diet from saturated to an n-3 PUFA or a low-fat diet significantly decreased the total cumulative energy intake compared with mice that continued the saturated fat diet [528 ± 19, 448 ± 13, and 593 ± 11 kJ · 4 wk-1 · mouse-1, respectively; F(2,14) = 85.26, P < 0.001, Fig. 2C]. Compared with the last week on the previous diet, there were significant reductions in the highly saturated to low-fat (HS-LF, -12%) and HS to n-3 PUFA (HS-n3, -16%) groups compared with the group continuing on the HS diet (Fig. 2D). Compared with the group continuing on the HS diet, the HS-n3 group had a small but significant reduction in caloric intake in weeks 8, 9, 10, and 11 (-21, -10, -6, and -8%, respectively). A more profound reduction was seen in the group switched to LF diet in weeks 8, 9, 10, or 11 (-18, -25, -28, and -27%, respectively). During the reversal phase, the rate of body weight gain was reduced in the HS-n3 group but was not statistically different between the HS and HS-LF groups (Fig. 3B).


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 2.   Total cumulative energy intake over 1 (A) and 7 (B) wk on the diets and for the 4 subsequent wk of dietary reversal (C). Changes of energy intake (D) were compared after 1 wk of dietary reversal to the last week on previous diet. Diets were, as described in MATERIALS AND METHODS, highly saturated (HS), n-6 PUFA (n-6), high n-3 PUFA (n-3), or low-fat (LF). P <=  0.05 vs. LF diet; P <=  0.01 vs. HS fat diet; P <=  0.05 vs. HS-LF.



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 3.   Food intake (A) and body wt (B) in mice fed an HS, n-3, n-6, or LF diet; or mice fed a high n-3 PUFA (HS-n3) or an LF (HS-LF) diet for 4 wk after 7 wk of an HS diet.

Body fatness. Relative body fatness was presented as body fat index, estimated as the amount of epididymal, perirenal, and inguinal fat depots per 100 g of body weight (Fig. 4). After 1 wk of diet, no significant differences were found in the body fat index between groups fed a highly saturated, n-6 PUFA, or low-fat diet (2.13 ± 0.08, 1.77 ± 0.22, 1.84 ± 0.10, respectively). However, the n-3 PUFA group had a significantly lower body fat index compared with each of the three other groups [1.28 ± 0.06, F(3,45) = 7.17, P < 0.001, Fig. 4A]. After 7 wk of diet, the mice fed a highly saturated fat diet had the highest relative body fat index (4.75 ± 0.29), significantly higher (P < 0.01) than n-3 PUFA and low-fat diet groups, where the latter two groups differed [n-3 PUFA: 2.37 ± 0.21, LF: 3.32 ± 0.35, F(3,35) = 8.65, P < 0.01, Fig. 4B]. Again, no difference was found in the body fat index between the n-6 PUFA and low-fat diets. Four-week dietary reversal showed that using a low-fat diet to replace the saturated fat diet did not significantly reduce the body fatness (group changed to low-fat diet: 5.11 ± 0.41; group continuing the saturated fat diet: 6.11 ± 0.55; Fig. 4C); however, changing to the n-3 PUFA diet did reduce it [2.16 ± 0.25, -64%, F(2,33) = 38.20, P < 0.01].


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 4.   Body fat index was measured after 1 (A) and 7 (B) wk of diet and 4 wk of dietary reversal (C). Mice were fed HS, n-6, n-3, or LF diets. aP <=  0.05 vs. LF diet; bP <=  0.05 vs. HS diet; cP <=  0.001 vs. HS-LF.

Plasma leptin. After 1 wk of diet, no statistical differences were found in the levels of plasma leptin among groups (Fig. 5A). After 7 wk of diet, a significant increase in plasma leptin was found in mice fed the saturated fat diet [4.8 ± 0.5 ng/ml, F(3,35) = 9.53, P < 0.001] compared with the mice fed the n-3 PUFA, n-6 PUFA, or low-fat diet (2.3 ± 0.1, 3.9 ± 0.1, 1.6 ± 0.2 ng/ml, respectively, Fig. 5B). Four weeks of dietary reversal showed that changing from a saturated to an n-3 PUFA fat diet resulted in a significant decrease in plasma leptin to a level comparable to that of the low-fat diet group [Fig. 3C, F(2,26) = 4.07, P = 0.029]. However, the group switched to low fat were intermediate and not different from the saturated fat group. Furthermore, plasma leptin levels correlated well with the body fat index after 7 wk of diets (r = 0.42, P = 0.007) and 4 wk of diet reversal (r = 0.34, P = 0.042), but not after 1 wk of diet (r = 0.12, P = 0.49).


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 5.   Levels of plasma leptin (ng/ml) were measured after 1 (A) and 7 (B) wk of diets and 4 wk of dietary intervention (C). Mice were fed HS, n-6, n-3, or LF diets. aP <=  0.05 vs. LF diet; bP <=  0.05 vs. HS diet; cP <=  0.05 vs. HS-LF.

Leptin receptor mRNA expression. After 1 wk of diet, no significant difference was found in the level of leptin receptor mRNA expression in the choroid plexus, Arc, or ventromedial hypothalamic nucleus (VMH) across diet groups (Fig. 6, A, B, and C). After 7 wk of diet, the saturated fat diet group showed a significant increase (+26, +21, and +30%) in the levels of leptin receptor mRNA expression in the choroid plexus, Arc, and VMH compared with the low-fat diet group (Fig. 6, A, B, and C). However, all of these increases in the saturated fat diet group disappeared after 11 wk of diet (not shown). In the VMH, leptin receptor mRNA expression was significantly lower in mice fed an n-6 and an n-3 PUFA diet than in mice fed the saturated fat diet (Fig. 6C). Changing the diet from a saturated to an n-3 PUFA or a low-fat diet for 4 wk had no significant effects on the leptin receptor mRNA expression in choroid plexus, Arc, or VMH (Fig. 6D).


View larger version (36K):
[in this window]
[in a new window]
 
Fig. 6.   Levels of leptin receptor mRNA expression were measured in choroid plexus (ChP, A), arcuate hypothalamic nucleus (Arc, B), and ventromedial hypothalamic nucleus (VMH, C). Mice were fed HS, n-6, n-3, or LF diets. Changing diet from HS to n-3 or LF had no significant effect on leptin receptor mRNA expression in ChP, Arc, or VMH (D). aP <=  0.05 vs. LF diet; bP <=  0.05 vs. HS diet.

NPY mRNA expression. After 1 wk of diet, the saturated fat diet group had a significantly reduced level of Arc NPY mRNA expression compared with the n-6 PUFA, n-3 PUFA, and low-fat diet groups (-47, -50, and -54%, respectively, Fig. 7A). No significant differences were found in Arc NPY mRNA expression among the latter three groups. After 7 wk of diet, the highly saturated fat diet group showed even further reduced Arc NPY mRNA expression compared with high n-3 PUFA, n-6 PUFA, and low-fat diet groups (to -50, -60, and -71%, respectively, Fig. 7B). Again, no significant differences were seen among the latter three groups. Changing the diet from the saturated to an n-3 PUFA or a low-fat diet significantly reversed Arc NPY mRNA expression (by +441 and +346%) compared with keeping mice on the saturated fat diet (Fig. 7C). Continued feeding of mice with the saturated fat diet led to an even more pronounced reduction, to <25% of Arc NPY mRNA expression when compared with the low-fat diet group. The reduction in NPY mRNA expression was fairly specific to the Arc. We have also quantified the levels of NPY mRNA expression in the primary motor cortex, primary sensory cortex, and amygdala. No dietary effects were observed in these areas at any time point (data not shown here).


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 7.   Levels of neuropeptide Y mRNA expression were measured after 1 wk (A), 7 wk (B), and 4 subsequent wk of dietary intervention (C). Results are shown for mice fed HS, n-6, n-3, or LF diets, and, after intervention, HS-LF and HS-n3 diets. aP <=  0.01 vs. LF diet.

AgRP mRNA expression. After 1 wk of diet, a significant reduction of Arc AgRP mRNA was found in mice fed the saturated fat diet (-21, -20, and -27%, Fig. 8A) compared with mice fed the n-6 PUFA, n-3 PUFA, or low-fat diet. No significant differences were found in the levels of Arc AgRP mRNA expression among the latter three groups. After 7 wk of diet, the saturated fat diet group still showed a reduced Arc AgRP mRNA expression (-23, -13, and -20%, Fig. 8B) compared with n-3 PUFA, n-6 PUFA, and low-fat diet groups. Again, no significant differences were found in the levels of Arc AgRP mRNA expression among the latter three groups. Four weeks of diet reversal showed that substitution of the saturated with the n-3 PUFA or low-fat diet had profound effects on Arc AgRP mRNA expression. Changing the diet from the saturated to a low-fat or a high n-3 PUFA fat diet significantly reversed Arc AgRP mRNA expression, by +181 and +237%, respectively, compared with mice staying on the saturated fat diet. Continued feeding of mice with the saturated fat diet led to an even more pronounced reduction, to <27% of Arc AgRP mRNA expression when compared with the low-fat diet group.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 8.   Levels of agouti-related peptide mRNA expression were measured after 1 wk (A), 7 wk (B), and 4 wk of dietary intervention (C). Results are shown for mice fed HS, n-6, n-3, LF, HS-LF, and HS-n3 diets. aP <=  0.05 vs. LF diet nCi/g tissue.

POMC mRNA expression. We have also measured Arc POMC mRNA expression in mice fed different types of fat and low-fat diets. We found no significant differences in the levels of POMC mRNA expression at any time points. The results are in the order of saturated, n-6 PUFA, n-3 PUFA, and low-fat diets for 1, 7, and 4 wk of dietary interventions. At 1 wk: 499 ± 56, 712 ± 85, 488 ± 40, 551 ± 31; at 7 wk: 446 ± 61, 402 ± 43, 457 ± 50, 414 ± 40; at 4 wk of dietary intervention: 391 ± 61, 310 ± 33, 273 ± 27, 328 ± 36 nCi/g tissue.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study has examined hypothalamic NPY, AgRP, POMC, and leptin receptor mRNA expression in response to high-fat diets emphasizing either saturated fat with a high n-6/n-3 PUFA ratio or PUFAs of either the n-3 or n-6 families. The main findings of this study are that a highly saturated fat diet potently decreases Arc NPY and AgRP mRNA expression and that this decrease occurs before significant elevations of circulating leptin levels can be detected. In contrast, diets equally high in total fat, but in which that fat is more predominantly PUFA, do not alter Arc NPY or AgRP mRNA expression levels compared with a low-fat diet. The decreased Arc NPY and AgRP mRNA expression levels induced by a highly saturated fat diet can be reversed by substitution of the diet with either high n-3 PUFA or low fat. POMC mRNA expression is not affected by dietary fat level or type, at least within the time frame studied.

A good deal of the research investigating the influence of diet on hypothalamic NPY mRNA expression and protein levels has focused on the balance between carbohydrate and fat. However, there are intertwined issues here beyond just macronutrient exchange. These include altered energy density, increased total caloric intake, and changes in body weight and fatness. The current study allows an instructive dissection of these issues.

The reduction in Arc NPY mRNA with obesogenic high-fat feeding found here has been reported from a number of laboratories, including our own (5, 6, 9, 23, 25). Arc NPY protein levels follow mRNA levels down with longer feeding periods. As well, NPY protein in the paraventricular hypothalamic nucleus falls with long-term feeding and development of obesity (23). The argument has then been made about the carbohydrate-to-fat ratio as a controller of hypothalamic NPY. Compared with a high n-3 fat diet, the current study shows that a high-fat diet with a higher proportion of saturated fat perturbs the NPY (and AgRP) system. This study suggests that it is not the level of dietary fat per se that influences NPY expression, but that it is some obesogenic property or properties of saturated fats, which are detected either peripherally or centrally. The NPY/AgRP systems then could be seen to be reacting in a homeostatic manner. In the case of the PUFA diets, there is no evidence for increased body fatness (and even less fatness in the case of the n-3 PUFA diet; see Fig. 4), and the lack of change in Arc NPY mRNA (or even slight rise by week 7 in the n-3 group) is appropriate. The issue is clearly not dietary fat vs. carbohydrate.

The homeostatic response (inhibition) of Arc NPY and AgRP mRNA expression in the highly saturated fat group should then, at least as one important factor, have been associated with hypophagia and have worked to normalize weight (and fat) gain. That is clearly only partially the case. If we first look at the high saturated fat vs. low fat comparison, then we see the effects of both fat type and increased dietary energy density. At 1 and 7 wk of feeding, Arc NPY and AgRP mRNA expressions are reduced in the highly saturated fat group by some 47 and 50% and 21 and 23%, respectively, compared with the low-fat group. However, despite these reductions, food intake was increased by 17 and 14%, and rate of body weight gain was also significantly higher in the highly saturated fat diet group compared with the low-fat group.

Comparisons among the high-fat diet groups allow us to separate out the effect of diets of higher caloric density. Here it can be seen that, because the low-fat and PUFA diets were similar in terms of NPY mRNA expression, there is again a substantial reduction (approx 50%) in the highly saturated fat group compared with the PUFA groups. However, in this situation, there is very small, but significant, reduction in food intake of some (e.g., approx 15% over weeks 4-7). This may indicate that, when caloric density is controlled, indeed downregulation of the NPY and/or AgRP systems does act to reduce intake. However, the effects are slow to take effect, extremely weak in relation to the size of the downregulation, and are more a small speed-bump than a roadblock on the highway to obesity.

These data then would seem to show that, indeed, the NPY and AgRP systems are reacting to some signal indexing positive energy balance (driven by the highly saturated fat diet) and are reacting appropriately. What is equally apparent is that the reduced Arc NPY and AgRP are insufficient to maintain energy homeostasis. Importantly, it is not high-fat diets per se that have this effect. High-fat diets emphasizing PUFAs do not act to impair energy balance, and the Arc NPY or AgRP systems are appropriately not responding. This strongly argues for a dysregulation in other parts of the energy balance system induced specifically by saturated fat, or at least by the fatty acid profile of that diet. Leptin is a potent regulator of the NPY system and one such candidate. However, at week 1, when Arc NPY mRNA levels were reduced by one-half, no increase in circulating leptin and no change in leptin receptor mRNA expression could be detected. If altered leptin dynamics were to account for the change in NPY, then a fairly subtle alteration in the 24-h profile would have to be implicated. This is possible but unlikely. Furthermore, if leptin had caused downregulation of Arc NPY, it should elevate Arc POMC mRNA as well (15, 19). However, this is not the case in this study, as there were no differences in POMC mRNA expression after 1 wk of diet. The conclusion that leptin is not responsible for the decline in NPY with fat feeding supports earlier suggestions (23). Other regulatory mechanisms must therefore exist in the regulation of Arc NPY and AgRP mRNA expression in the mice fed a highly saturated fat diet.

One of the possible explanations for the effects of the different high-fat diets is specific fatty acid modulation of gene expression. Clarke and coworkers (2, 3) showed very early that fatty acid synthase (FAS) gene expression was increased by diets high in saturated and monounsaturated fats, but that when the predominant fat was n-6 PUFA, FAS activity was lower, and when it was n-3 PUFA, FAS activity was returned almost to control level. Because endogenous lipogenesis creates saturated fats, an increased FAS activity will result in a "saturating down" of the whole body fatty acid pool, adding to the overall effect of saturated fats. This major increase in the body's saturated fatty acid pool may then directly result in obesity via decreases in metabolic rate (e.g., reduced ion "leakiness of membrane and reduced adrenergic binding"; see Refs. 12 and 22). Equally, the effects may be indirect. Genes implicated in the mature phase of adipocyte proliferation are highly expressed in rats fed saturated and monounsaturated fats (21). In contrast, expression was suppressed to some extent by n-6 PUFAs and profoundly by n-3 PUFAs. There are numerous secreted proteins from adipocytes besides leptin, and one of these, which is primarily expressed in the transition phase from pre- to mature adipocyte, might potently regulate NPY and associated neuropeptides. Of course, although the effects of different dietary fatty acid profile might be on gene expression in adipose and other peripheral tissues, a direct effect on brain is also possible. This exciting possibility might be addressed in future work with brain slice preparations.

An important aspect of the current studies is the effect seen with the dietary reversal phase. Both change in dietary fat profile (n-3 diet) and alteration in fat/carbohydrate balance (i.e., low-fat diet) had beneficial effects on body fatness. However, it is striking that these effects are very modest with the low-fat diet, compared with major effects with change only in dietary fat profile. This is equally reflected in the leptin levels. In contrast, both dietary reversal interventions resulted in "normalization" (to the chronic low-fat control) of NPY and AgRP mRNA expression. This again suggests the possibility of direct effects peripherally on gene expression (in adipose tissue) of individual fatty acids, which tune metabolism away from storage and toward mobilization of triglycerides. An analysis of adipocyte gene expression (e.g., lipoprotein lipase, perilipin, hormone-sensitive lipase, and the like) and of fatty acid efflux in the early period after dietary reversal intervention would be informative.

The lack of effect of highly saturated fat diet on POMC expression over the current dietary time frame is consistent with our earlier work (10) and that of others (8). The current results extend this observation by showing that neither fat/carbohydrate balance nor dietary fatty acid profile has effects. Whatever the factor or factors are that alter NPY/AgRP expression, they are not effective on the POMC system. Finally, a limitation of the current study is that the observed changes in NPY gene expression may not precisely reflect NPY release. It is possible that dietary fat profile might somehow differentially affect cell membrane phospholipid composition and protein secretion and interaction. This possibility should be tested in future studies.

In summary, the current studies demonstrate that changing the fatty acid profile of the diet alone can profoundly alter the expression of major hypothalamic neuropeptides of energy balance. The effects would appear to relate to whether a particular dietary fatty acid profile is obesogenic or not, but dysregulation appears independent of leptin as a mediator. The next step is to understand the elements of the body energy balance homeostatic network, which are dysregulated by saturated fats.


    ACKNOWLEDGEMENTS

We thank Associate Professors A. Jenkins and P. McLennan for helpful discussion and suggestions during this study, and Warren Bell for helpful reading of the manuscript. We also acknowledge R. P. Scherer of Australia for supplying n-3 oil (MaxEPA) for this study.


    FOOTNOTES

This study was supported by a grant from the National Health and Medical Research Council of Australia of Australia.

Address for reprint requests and other correspondence: X.-F. Huang, Dept. of Biomedical Science, Univ. of Wollongong, NSW 2522, Australia (E-mail: Xu_Feng_Huang{at}uow.edu.au).

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/ajpendo.00230.2001

Received 29 May 2001; accepted in final form 23 February 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Beck, B. Neuropeptides and obesity. Nutrition 16: 916-923, 2000[ISI][Medline].

2.   Clarke, S, Baillie R, Jump D, and Nakamura M. Fatty acid regulation of gene expression: its role in fuel partitioning and insulin resistance. Ann NY Acad Sci 827: 178-186, 1997[Abstract].

3.   Clarke, SD, Turini M, and Jump D. Polyunsaturated fatty acids regulate lipogenic and peroxisomal gene expression by independent mechanisms. Prostaglandins Leukot Essent Fatty Acids 57: 65-69, 1997[ISI][Medline].

4.   Erickson, JC, Hollopeter G, and Palmiter RD. Attenuation of the obesity syndrome of ob/ob mice by the loss of neuropeptide Y. Science 274: 1704-1707, 1996[Abstract/Free Full Text].

5.   Giraudo, SQ, Kotz CM, Grace MK, Levine AS, and Billington CJ. Rat hypothalamic NPY mRNA and brown fat uncoupling protein mRNA after high-carbohydrate or high-fat diets. Am J Physiol Regulatory Integrative Comp Physiol 266: R1578-R1583, 1994[Abstract/Free Full Text].

6.   Guan, XM, Yu H, Trumbauer M, Frazier E, Van der Ploeg LH, and Chen H. Induction of neuropeptide Y expression in dorsomedial hypothalamus of diet-induced obese mice. Neuroreport 9: 3415-3419, 1998[ISI][Medline].

7.   Hahn, TM, Breininger JF, Baskin DG, and Schwartz MW. Coexpression of Agrp and NPY in fasting-activated hypothalamic neurons. Nat Neurosci 1: 271-272, 1998[ISI][Medline].

8.   Harrold, JA, Williams G, and Widdowson PS. Changes in hypothalamic agouti-related protein (AGRP), but not alpha-MSH or pro-opiomelanocortin concentrations in dietary-obese and food-restricted rats. Biochem Biophys Res Commun 258: 574-577, 1999[ISI][Medline].

9.   Lin, S, Storlien LH, and Huang XF. Leptin receptor, NPY, POMC mRNA expression in the diet-induced obese mouse brain. Brain Res 875: 89-95, 2000[ISI][Medline].

10.  Lin S, Thomas TC, Storlien LH, and Huang XF. Development of obesity and central leptin resistance in high-fat diet induced obese mice. Int J Obes Rel Metab Disorders: 1-8, 2000.

11.   Marsh, DJ, Hollopeter G, Huszar D, Laufer R, Yagaloff KA, Fisher SL, Burn P, and Palmiter RD. Response of melanocortin-4 receptor-deficient mice to anorectic and orexigenic peptides. Nature Genet 21: 119-122, 1999[ISI][Medline].

12.   Matsuo, T, Sumida H, and Suzuki M. Beef tallow diet decreases beta-adrenergic receptor binding and lipolytic activities in different adipose tissues of rat. Metabolism 44: 1271-1277, 1995[ISI][Medline].

13.   Mercer, JG, Lawrence CB, and Atkinson T. Regulation of galanin gene expression in the hypothalamic paraventricular nucleus of the obese Zucker rat by manipulation of dietary macronutrients. Brain Res Mol Brain Res 43: 202-208, 1996[ISI][Medline].

14.   Mercer, JG, Moar KM, Rayner DV, Trayhurn P, and Hoggard N. Regulation of leptin receptor and NPY gene expression in hypothalamus of leptin-treated obese (ob/ob) and cold-exposed lean mice. FEBS Lett 402: 185-188, 1997[ISI][Medline].

15.   Mizuno, TM, Kleopoulos SP, Bergen HT, Roberts JL, Priest CA, and Mobbs CV. Hypothalamic pro-opiomelanocortin mRNA is reduced by fasting in ob/ob and db/db mice, but is stimulated by leptin. Diabetes 47: 294-297, 1998[Abstract].

16.   Mizuno, TM, and Mobbs CV. Hypothalamic agouti-related protein messenger ribonucleic acid is inhibited by leptin and stimulated by fasting. Endocrinology 140: 814-817, 1999[Abstract/Free Full Text].

17.   Naveilhan, P, Hassani H, Canals JM, Ekstrand AJ, Larefalk A, Chhajlani V, Arenas E, Gedda K, Svensson L, Thoren P, and Ernfors P. Normal feeding behavior, body weight and leptin response require the neuropeptide Y Y2 receptor. Nat Med 5: 1188-1193, 1999[ISI][Medline].

18.   Schwartz, MW, Baskin DG, Bukowski TR, Kuijper JL, Foster D, Lasser G, Prunkard DE, Porte DJ, Woods SC, Seeley RJ, and Weigle DS. Specificity of leptin action on elevated blood glucose levels and hypothalamic neuropeptide Y gene expression in ob/ob mice. Diabetes 45: 531-535, 1996[Abstract].

19.   Schwartz, MW, Seeley RJ, Woods SC, Weigle DS, Campfield LA, Burn P, and Baskin DG. Leptin increases hypothalamic pro-opiomelanocortin mRNA expression in the rostral arcuate nucleus. Diabetes 46: 2119-2123, 1997[Abstract].

20.   Shutter, JR, Graham M, Kinsey AC, Scully S, Luthy R, and Stark KL. Hypothalamic expression of ART, a novel gene related to agouti, is up-regulated in obese and diabetic mutant mice. Genes Dev 11: 593-602, 1997[Abstract].

21.   Storlien, L, Huang X, Lin S, Xin X, Wang H, and Else P. Dietary fat subtypes and obesity. In: Fatty Acids and Lipids---New Findings, edited by Hamazaki T, and Okuyama H. Basel: Karger, 2001, p. 148-154.

22.  Storlien LH, Hulbert AJ, and Else PL. Polyunsaturated fatty acids, membrane function and metabolic diseases such as diabetes and obesity. Curr Opin Clin Nutr Metab Care: 559-563, 1998.

23.   Stricker Krongrad, A, Cumin F, Burlet C, and Beck B. Hypothalamic neuropeptide Y and plasma leptin after long-term high-fat feeding in the rat. Neurosci Lett 254: 157-160, 1998[ISI][Medline].

24.  Wang HQ, Storlien L, and Huang XF. Influence of dietary fats on c-Fos-like immunoreactivity in mouse hypothalamus. Brain Res: 184-192, 1999.

25.   Wang, J, Akabayashi A, Dourmashkin J, Yu HJ, Alexander JT, Chae HJ, and Leibowitz SF. Neuropeptide Y in relation to carbohydrate intake, corticosterone and dietary obesity. Brain Res 802: 75-88, 1998[ISI][Medline].

26.   Wang, Q, Bing C, Al Barazanji K, Mossakowaska DE, Wang XM, McBay DL, Neville WA, Taddayon M, Pickavance L, Dryden S, Thomas ME, McHale MT, Gloyer IS, Wilson S, Buckingham R, Arch JR, Trayhurn P, and Williams G. Interactions between leptin and hypothalamic neuropeptide Y neurons in the control of food intake and energy homeostasis in the rat. Diabetes 46: 335-341, 1997[Abstract].

27.   Zhang, Y, Proenca R, Maffei M, Barone M, Leopold L, and Friedman JM. Positional cloning of the mouse obese gene and its human homologue. Nature 372: 425-432, 1994[ISI][Medline].


Am J Physiol Endocrinol Metab 282(6):E1352-E1359
0193-1849/02 $5.00 Copyright © 2002 the American Physiological Society