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Ingestive Behavior and Neurosciences

Dietary Fat Concentration Influences the Effects of trans-10, cis-12 Conjugated Linoleic Acid on Temporal Patterns of Energy Intake and Hypothalamic Expression of Appetite-Controlling Genes in Mice^1–3,

Food and Nutritional Science Division, School of Biological Sciences, The University of Hong Kong, Hong Kong, People's Republic of China

^* To whom correspondence should be addressed. E-mail: etsli{at}hku.hk.

	ABSTRACT

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
This study tested the hypothesis that the effect of trans-10, cis-12 conjugated linoleic acid (t10, c12 CLA) on energy intake (EI) and body weight (BW)/composition is confounded by dietary fat concentration and involves hypothalamic appetite-controlling mechanisms. ICR mice received low-fat (LF; 5 g/100 g) or high-fat (HF; 30 g/100 g) diets, with or without 0.5 g/100 g t10, c12 CLA (>98% pure) for 27 d. By d 13, BW and cumulative EI of the mice fed CLA supplemented LF diet (LF/CLA) were 6.6 and 23.6% lower, respectively, than the LF mice. In the subsequent 14 d, their EI rebounded and did not differ from the LF group. BW and EI did not differ between the HF and CLA supplemented HF (HF/CLA) groups. Hypothalamic pro-opiomelanocortin (POMC) mRNA expression was elevated (P = 0.031) on d 13 but suppressed (P < 0.001) on d 27 due to CLA treatment. CLA also suppressed AMP-activated protein kinase 2 expression. Mice in Expt. 2 received the LF diet, the LF/CLA, or were pair-fed the LF diet to the EI of the CLA group (LF/PF). LF/CLA and LF/PF mice did not differ in the hypothalamic POMC:neuropeptide Y expression ratio on d 13, but it was significantly lower in the LF/PF group on d 27. We conclude that the habitual dietary fat concentration influences the magnitude of weight loss induced by dietary t10, c12 CLA. The effect is in part independent of EI. Hypothalamic neuropeptides and nutrient sensing mechanisms may play a role.

	Introduction

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

It is generally acknowledged that CLA induced body composition changes (4) and, specifically, the trans-10, cis-12 isomer (t10, c12 CLA) is responsible for a reduction in body fat in mice (5). However, the mechanism by which this effect occurs remains unknown. The isomer can impair differentiation (6–8) and initiate apoptosis of white adipocytes (9–11). Reduction in adiposity is also associated with increased hepatic fatty acid oxidation (12,13), energy expenditure (14–16), and energy loss in excreta (15). In contrast to the peripheral/metabolic effects, studies examining the involvement of changes in energy intake (EI) have had mixed results. Appetite in humans was not affected (17) and nonsignificant change in EI was reported in mice (14,15,18–20). However, others observed a significant reduction in EI (5,10). These discrepancies likely arose from differences in the composition, dosage, and duration of CLA treatment. For instance, most of the earlier studies used CLA mixtures containing both c9, t11 and t10, c12 isomers. This is further complicated by nonstandardized habitual diets, exemplified in a study demonstrating that the extent of CLA-mediated lipodystrophy is influenced by the total fat content of the diet (21).

If EI is altered by CLA, it would be important to determine the involvement of brain mechanisms that affect appetite. Because CLA isomers are incorporated and metabolized in rodent brain (22), it is of interest to note that intracerebroventricular injection of a CLA mixture suppressed mRNA expression of neuropeptide Y (NPY) and agouti-related protein and caused food intake suppression (23). These orexigenic peptides can stimulate feeding (24) and act antagonistically to the anorexigenic pro-opiomelanocortin (POMC) (25,26). In addition, AMP-activated protein kinase (AMPK), the nutrient-sensing mechanism (27,28), might be involved. High-protein diet–induced weight loss has been associated with reduced AMPK activity but enhanced POMC expression (29). The effect of dietary CLA on brain appetite mechanisms has not been determined.

This study aimed to test the hypothesis that the effect of t10, c12 CLA on EI and body weight (BW)/composition is confounded by dietary fat content and involves hypothalamic appetite-controlling mechanisms. The temporal feeding pattern was monitored in ICR mice fed low-fat (LF) or high-fat (HF) diets with or without t10, c12 CLA supplementation. We used a pair-feeding approach in the Expt. 2 to further delineate the appetite-suppressing effect of this isomer. These behavioral changes were interpreted in the context of hypothalamic mRNA expressions of POMC, NPY, and AMPK2 catalytic subunit.

	Materials and Methods

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
Mice and diets

Male ICR mice were obtained from the Laboratory Animal Unit, University of Hong Kong. All animal protocols were approved by the university's committee on the use of living animals in teaching and research. Mice were house individually and kept in a controlled environment at 22°C under a 12-h-light/12-h-dark cycle with light on from 0700 to 1900. Mice consumed ad libitum water and laboratory food (Lab Diet, The Richmond Standard, PMI Nutritional International) for 1 wk before receiving the experimental diets. The AIN-93G–based (30) semipurified diets were modified to contain 50 g corn oil/kg diet as LF (5% fat, wt:wt) diet and 300 g fat/kg diet as HF (30% fat, wt:wt) diet. The additional fat was added at the expense of the cornstarch component. When t10, c12 CLA (>98% pure; Matreya) was added, it replaced the corn oil and at a fixed concentration of the diet (0.5 g/100 g) (diet compositions in Table 1). All prepared diets were stored at –20°C and fresh diets were provided 2 to 3 times weekly.

    Expt. 1: Effect of dietary fat. Mice (4 wk old; n = 49) were randomly assigned to the LF (n = 24) or HF (n = 25) diet group for 8 wk. Mice at each fat level were then further divided (based on BW) into 2 subgroups (n = 12 or 13 per group). Mice in the subgroups either continued to receive the original diet or were fed a diet supplemented with t10, c12 CLA. In a preliminary experiment, we observed a biphasic pattern on EI and weight gain; hence, some mice in each subgroup were killed after 13 d, whereas the remaining mice continued to be fed up to 27 d. BW was recorded regularly (in 0.1 g). Feed intake was measured as the disappearance (in 0.1 g) from spillage-proofed glass food cups equipped with a stainless steel, small-holed cover lid. On d 13 and 27, mice were killed after 12-h overnight food deprivation. Blood, organ tissues, and hypothalami were collected and immediately frozen in liquid nitrogen and stored at –80°C until analysis (31).

    Expt. 2: Pair-feeding. This experiment used only LF diets with or without CLA supplementation. Mice (11 wk old; n = 42) were randomly divided into 3 groups. The first 2 groups had free access to the LF diet or the LF diet with 0.5 g/100 g CLA (LF/CLA). Mice in the 3rd group were fed the LF diet but pair-fed to the mean EI of the LF/CLA group on the previous day (LF/PF).

RNA extraction and RT-PCR

RNA of mouse hypothalamus was isolated using Trizol reagent (Invitrogen, cat. no.: 15596–018) and first-strand cDNA was synthesized using the AMV RT system (A3500, Promega) according to the manufacturer's protocol. For each hypothalamus, the RNA pellet was redissolved in 20 µL RNase-free water. Then 1 µg RNA was loaded for RT in each 10-µL reaction mix. The resultant cDNA was diluted with RNase-free water and 1 µL of the diluted cDNA was applied in 25 µL PCR, with appropriate annealing temperatures and amplification cycles. Target genes under investigation and the corresponding primers used are shown in Supplemental Table 1.

Five microliters of the PCR product per sample was loaded into 1.5% agarose gel for gel electrophoresis. Bands were visualized by precasting the gel using SYBR Saf DNA gel stain (Invitrogen, cat. no.: MP33100) with the appropriate concentration following the manufacturer's protocol. Band intensities were detected and analyzed by ImageJ 1.37v software.

Statistical analysis

All data were expressed as means ± SEM. SPSS 12.0 for Windows was used as statistical software. For Expt. 1, the BW of the LF and HF mice at the end of the habituation period was compared by t test. The effects of dietary fat level and CLA supplementation were analyzed by 2-way ANOVA followed by Duncan's multiple range tests for group comparisons if the interaction was significant. For each time period, BW at the beginning and total EI served as covariate in the analyses of EI and BW, respectively. For Expt. 2, Duncan's multiple comparisons were performed if the main effect was significant as determined by 1-way ANOVA. For Tables 2 and 4, all mice were included to generate EI and BW data of the first 13 d. For d 14–27, the number of mice was reduced by a one-half, because 6 or 7 mice in each dietary group were killed on d 13. We used paired t tests to compare EI/BW between d 1–13 and d 14–27 and included only those mice that completed the entire study period. These mice were used to generate plots in Figure 1. Within each day, EI and BW of the LF/CLA and LF/PF groups were compared with the LF group using Dunnett's test. Gene expression was presented as relative mRNA, which is the β-actin normalized densitometric value. Two-tailed tests were considered significant at P < 0.05.

View larger version (20K): [in this window] [in a new window]   FIGURE 1  Food intake (A) and BW (B) of ICR mice fed a LF diet with or without CLA or pair-fed the LF diet to the mean intake of the mice fed CLA for 27 d (Expt. 2). Values are means ± SEM, n = 7 or 8. #Different from both other groups at that time; *different from LF at that time, P < 0.05.

	Results

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

    EI and BW. After the habituation period, the BW gain of the HF mice was 15.8% higher than that of the LF mice (P < 0.05). EI from d 1–13 was affected by dietary fat (P = 0.027), CLA (P < 0.001), and their interaction (P < 0.001). The EI of the LF/CLA group was significantly less than that of the other 3 groups, which did not differ. The EI of the LF/CLA mice was 23.6% lower than that of the LF mice. BW gain was affected by dietary fat and CLA (P < 0.001). From d 1 to 13, BW lost (2.2 g) due to CLA was significant only in the LF/CLA group (P = 0.023; Table 2).

The treatments did not affect EI between d 14 and 27 (Table 2). However, the final BW of the LF groups was lower than that of the HF groups (P = 0.01); the former groups actually gained more than the latter groups during this time period (P = 0.008). To investigate the relationship between EI and BW, EI was expressed on a per-gram–BW basis and compared between the 2 time periods within each subgroup of those mice that completed the entire period. As the mice got older, EI/BW between d 14–27 were lowered than that of d 1–13 (LF, P < 0.01; HF, P < 0.001). However, the LF/CLA mice experienced a rebound in EI/BW between d 14–27 (26.5 ± 0.9 kJ/g) compared with d 1–13 (21.9 ± 1.0 kJ/g; P < 0.001), whereas it remained unchanged in the HF/CLA mice (26.0 ± 0.6 kJ/g vs. 26.5 ± 0.7 kJ/g, respectively) (Table 2).

    Hypothalamic mRNA expression. Overall, CLA treatment increased the mRNA expression of POMC on d 13 (P < 0.05). Neither dietary fat nor CLA significantly affected NPY mRNA expression (Table 3). The dietary fat level tended to affect the POMC:NPY mRNA ratio (P = 0.054), with higher values in the mice fed the LF diet. CLA supplementation reduced AMPK2 mRNA (P < 0.05).

View this table: [in this window] [in a new window]   TABLE 3 NPY, POMC, AMPK2, and MC4R mRNA expressions in hypothalamus of mice fed LF or HF diet with or without t10, c12 CLA (Expt. 1)1

Expt. 2

    EI and BW. Similar to Expt. 1, the EI of LF/CLA mice during the first 13 d was 29.6% less than that of the LF control (Fig. 1A; Table 4). On d 13, BW of the LF/PF group was less than that of the LF/CLA group (P < 0.05).

Due to a delay in removing food cups on d 14, food intake was higher than usual and as a consequence, recorded intake for d 15 was much lower. The mean of the 2 d did not differ from d 13 or 16.

Again, the CLA mice exhibited a rebound in EI between d 14–27. The EI/BW did not differ among the 3 groups. Compared with the first 13 d, EI/BW of the LF mice in the subsequent period was reduced by 11.7% (P < 0.01), whereas that of the CLA mice increased by 31% (P < 0.001). Despite similar EI in this period, weight gain differed between the LF/CLA (0.4 g) and LF/PF (2.8 g) mice (P < 0.001). BW of the 2 groups started to deviate on d 22 and the LF/PF group was heavier than the LF/CLA group on d 25–27 (P < 0.05; Fig. 1B). On d 27, visceral fat mass was lower in the CLA mice (0.2 g) than in the PF mice (1.7 g; P < 0.05; Table 4).

    Hypothalamic mRNA expression. On d 13, the mRNA expression of POMC and NPY was not affected by CLA or pair-feeding (Table 5). However, pair-feeding decreased the mRNA expression ratio of POMC:NPY compared with the LF group (P < 0.05). AMPK2 mRNA expression in the LF/PF mice was similar to that of the CLA mice but lower than that of the LF control (P < 0.05).

View this table: [in this window] [in a new window]   TABLE 5 NPY, POMC, AMPK2, and MC4R mRNA expressions in hypothalamus of mice fed a LF, LF/CLA, or LF/PF diet (Expt. 2)1

	Discussion

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
The habitual diet is a confounding factor in studying the impact of t10, c12 CLA supplementation on EI and BW composition of mice. Mice fed a HF diet receiving t10, c12 CLA did not gain weight, whereas LF mice lost over 3–6% of BW. Mice given CLA exhibited a biphasic response first with anorexia then followed by appetite rebound. Fluctuation in EI, however, is not a key attribute to changes in BW and body fat content, because the effects were not replicated in the LF/PF mice. Appetite fluctuations were associated with changes in the expressions of hypothalamic POMC, NPY, and AMPK2.

At the end of Expt. 1, t10, c12 CLA caused weight loss (–1.2 g) in the LF mice, but the HF/CLA mice maintained their initial BW (Table 2). These data explained the conflicting results of studies using only HF (14,15,20) or LF (5) diets. More importantly, the present study extends the observation to demonstrate a biphasic fluctuation in the BW of the LF/CLA mice of a substantial loss in the first 13 d followed by a modest regain in the next dietary period. These changes were accompanied by matching fluctuations in EI (see EI/BW in Table 2). Hence, t10, c12 CLA affected mice differently depending on the dietary fat concentration. Our results contrast those of an earlier observation reporting a larger decrease in EI in mice fed a HF diet than in those fed a LF diet (32). The discrepancy between these 2 studies likely arose from the differences in composition and dosage of the 2 CLA preparations. In the latter study, a mixture containing 40.7% t10, c12 CLA was given at a level of 1.2 and 1.0% by weight in the HF and LF diets, respectively.

The question of whether weight loss in CLA mice was entirely due to reduced EI was addressed using the pair-feeding approach (Expt. 2). Weight gain in the LF/PF mice was 7 times that of the LF/CLA mice between d 14 and 27. To our knowledge, this is the first study to demonstrate a suppressive effect of t10, c12 CLA on BW that is independent of EI. Furthermore, the difference in BW between the LF/CLA and LF/PF mice at the end of the study could be explained almost entirely by the difference in visceral fat mass. The data also corroborate with results of a recent study using the pair-feeding approach (33). Although the CLA-supplemented mice had higher BW, their percent body fat was lower than that of the pair-fed mice. The contradictory results might be related to the differences in methodology in which all mice were previously food restricted and a mixture of CLA isomer was used. Nevertheless, these results provide strong evidence for a regulatory role of t10, c12 CLA in lipid metabolism and that t10, c12 CLA can modulate BW and composition via mechanisms independent of EI. The present results, however, do not provide an explanation for the biphasic effect of t10, c12 CLA. This novel observation warrants further investigation.

The differential effects of t10, c12 CLA against a LF and HF background might be explained by the proportion of fat as CLA in the diet. Although both diets contained 0.5 g CLA/100 g, each 1 g of fat in the LF/CLA diet contained 0.1 g CLA, whereas that of the HF/CLA diet contained only 0.0167 g. If one interprets this as a "dilution effect," then this might be a biologically important response, because a higher amount of fat in a CLA-supplemented diet has been shown to reduce lipodystrophy in mice (21). Because apoptosis was implicated as one possible mechanism in reducing retroperitoneal fat mass (10), it is speculated that the dilution effect might be responsible in part for a weaker apoptotic effect in the HF/CLA mice.

We attempt to relate the appetite fluctuation with expression of hypothalamic appetite markers. In Expt. 1, regardless of dietary fat level, suppression of EI in CLA mice during the first 13 d was accompanied by an elevated mRNA expression of the anorexigenic POMC (Table 3). During the appetite rebound phase (d 14–27), an increase in NPY mRNA as well as a reciprocal decrease in POMC mRNA expression were observed (these changes were nonsignificant in Expt. 2). Because POMC and NPY have opposing effects on food intake regulation, the POMC:NPY mRNA ratio was calculated as an indicator of appetite change. CLA treatment significantly reduced the ratio on d 27. Others had employed the agouti-related protein:POMC mRNA ratio to reflect diurnal fluctuations in appetite (34). The POMC:NPY mRNA ratio appears to be rather sensitive, because the magnitude of decrease in this ratio after CLA feeding was associated with a different degree of appetite rebound, being greater for the LF/CLA mice than for the HF/CLA mice. Hence, the data suggest a possible involvement of the 2 neuronal systems in the temporal pattern of EI when mice were given t10, c12 CLA.

When EI was matched as in the LF/CLA and LF/PF groups of Expt. 2, the POMC:NPY mRNA ratio in the former was significantly higher than that of the latter group on d 27 (Table 5), suggesting the possible involvement of other neuronal systems. Although we could not observe any changes in the expression of MC4R, some interesting results were obtained with the mRNA expression of 2-catalytic subunit of AMPK. The expression was suppressed by CLA with the effect being independent of dietary fat concentration and more pronounced in the 2nd half in both experiments. Because stimulation of AMPK expression occurs in a low-energy state (28), our data suggest that an "energy-rich" state was interpreted by the CLA mice. A possible explanation is that CLA isomers are preferred substrates of metabolism/oxidation in all tissues, particularly the brain (22). Hence, a localized increase in cellular energy availability resulting from oxidation of t10, c12 CLA (22) might set the stage for reduced AMPK activity in conjunction with other network mechanisms.

This energy-rich state assumption could be taken further to suggest that AMPK expression might play a permissive role in governing BW changes in mice receiving t10, c12 CLA. In Expt. 2, BW rebounded in the LF/PF mice but not in the LF/CLA mice; the former gained 3.5% of their initial BW, whereas the LF/CLA mice had a 3.4% loss (Fig. 1B). Noticeably, the LF/PF mice had an AMPK2 expression level similar to the LF control mice, but that of the LF/CLA mice was suppressed. Because the 2 groups had similar EI, the data suggest that inhibition of the AMPK system might somehow be involved in the modulation of BW and composition.

It is acknowledged that the mRNA expression data herein only provide a snapshot of the complex mechanisms of how t10, c12 CLA affects food intake. Our results will benefit from additional analyses that track regional changes. For instance, immunofluorescence staining could reveal downstream changes in a specific site, such as the paraventricular nucleus, arcuate nucleus, and lateral hypothalamic area. Monitoring hypothalamic AMP and ATP in conjunction with information regarding CLA oxidation would also generate data to validate the energy-rich state presumption in the CLA-supplemented mice.

In summary, the effects of t10, c12 CLA depend on dietary fat concentration, being more pronounced in mice fed a LF diet. The modulatory effect of t10, c12 CLA on BW and body fat content is independent of reduced EI. The changes in mRNA expression of hypothalamic POMC and NPY together with that of the AMPK provide a new avenue for the exploration of the relationship between CLA, appetite-controlling mechanisms, and the regulation of body composition.

	ACKNOWLEDGMENTS

 
Special thanks to Dr. Laureen L. Y. Chan for technical support and assistance.

	FOOTNOTES

 
¹ Supported by the Faculty Development Fund.

² Author disclosures: M. H. H. So, I. M. Y. Tse, and E. T. S. Li, no conflicts of interest.

³ Supplemental Table 1 is available with the online posting of this paper at jn.nutrition.org.

⁴ Abbreviations used: AMPK, AMP-activated protein kinase; BW, body weight; CLA, conjugated linoleic acid; EI, energy intake; HF, high-fat diet; LF, low-fat diet; LF/CLA, LF diet with CLA; LF/PF, pair-fed the LF diet to the EI of the CLA group; MC4R, melanocortin 4 receptor; NPY, neuropeptide Y; POMC, pro-opiomelanocortin; t10, c12 CLA, trans-10, cis-12 conjugated linoleic acid.

Manuscript received 4 June 2008. Initial review completed 11 July 2008. Revision accepted 12 October 2008.

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