C75 Alters Central and Peripheral Gene Expression to Reduce Food Intake and Increase Energy Expenditure
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内分泌学杂志 2005年第1期
Departments of Neuroscience (Y.T., E.-K.K., G.V.R.), Pathology (J.N.T., M.L.P., F.P.K.), Psychiatry (T.H.M.), Neurology (G.V.R.), Biological Chemistry (F.P.K.), and Oncology (F.P.K.), The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
Address all correspondence and requests for reprints to: Dr. Gabriele V. Ronnett, Department of Neuroscience, 1006B Preclinical Teaching Building, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205. E-mail: gronnett@jhmi.edu.
Abstract
C75, a synthetic inhibitor of fatty acid synthase (FAS), causes anorexia and profound weight loss in lean and genetically obese mice. C75 also acts as a stimulator of carnitine palmitoyltransferase-1 to induce fatty acid oxidation. To approximate human obesity, we used a 2-wk C75 treatment model for diet-induced obese (DIO) mice to investigate the central and peripheral effects of C75 on gene expression. C75 treatment decreased food intake, increased energy expenditure, and reduced body weight more effectively in DIO than in lean mice. Analysis of the gene expression changes in hypothalamus demonstrated that the reduced food intake in C75-treated DIO mice might be mediated by inhibition of orexigenic neuropeptide expression and induction of anorexigenic neuropeptide expression. Gene expression changes in peripheral tissues indicated that C75 increased energy expenditure by the induction of genes involved in fatty acid oxidation. C75 also inhibited the expression of genes in peripheral tissues responsible for fatty acid synthesis and accumulation. The patterns of the changes in central and peripheral gene expression that occur with C75 treatment provide mechanisms to explain the reduced food intake and increased energy expenditure observed with C75.
Introduction
OBESITY HAS NOW surpassed tobacco as the leading cause of preventable mortality in the United States (1). Resulting from a chronic imbalance between energy intake and energy expenditure, obesity is frequently associated with other diseases, such as diabetes and hypertension (2, 3). Although appropriate diet and exercise may lead to a reduction in adipose mass, many patients eventually regain the weight (4). Thus, pharmacological treatment may be required for the control of obesity in many patients.
C75, a compound designed as an inhibitor of fatty acid synthase (FAS), causes reduced food consumption and increased fatty acid oxidation in diet-induced obese (DIO) mice, leading to profound loss of adipose tissue (5). Studies performed by us and others have shown that the anorexigenic effects of C75 are mediated through alterations of hypothalamic neuropeptide expression (5, 6, 7, 8, 9). C75 reduces neuropeptide Y (NPY) and agouti-related protein (AGRP) expression in lean mice in acute treatment experiments (5, 6, 7). We and others have also shown that C75 affects hypothalamic neuropeptide expression in lean and obese (ob/ob) mice in acute treatment experiments (9) and in lean, obese (ob/ob), and DIO mice in multiple-day treatment experiments (5, 8). Moreover, the central effect of C75 on the expression of hypothalamic neuropeptides is probably mediated through C75 modulation of neuronal ATP levels and ensuing alterations in AMP-activated protein kinase phosphorylation and activity (10). The increased fatty acid oxidation noted both in vitro and in vivo has been shown to be due at least in part to direct C75 stimulation of carnitine palmitoyltransferase-1 (CPT-1), the pace-setting enzyme of mitochondrial fatty acid oxidation (11, 12).
In addition to affecting hypothalamic neuropeptide expression, C75 could potentially alter the expression of genes involved in fatty acid metabolism due to its direct effects on fatty acid synthesis and oxidation. In this study we used a 2-wk chronic C75 treatment model suitable for both DIO and lean mice and examined the effects of C75 on body weight, food intake, and energy expenditure. We also quantified the expression of key genes involved in fatty acid metabolism in liver, white adipose tissue (WAT) and skeletal muscle along with hypothalamic neuropeptide expression.
Our results showed that the 2-wk C75 treatment was more efficacious in DIO mice than in lean mice, as evidenced by the increased weight loss, decreased food intake, and increased energy expenditure in DIO mice. In the hypothalamus of DIO mice, C75 treatment caused a marked anorexigenic neuropeptide profile, with reduction of orexigenic neuropeptide expression and increased anorexigenic neuropeptide expression. In WAT obtained from DIO mice, C75 treatment altered the expression of genes involved in fatty acid metabolism to favor fatty acid oxidation, without increasing the expression of peroxisome proliferator-activated receptor(PPAR). Uncoupling protein 2 (UCP2) expression was also increased in liver, WAT, and skeletal muscle of C75-treated DIO mice. Thus, in addition to direct effects on FAS and CPT-1 enzyme activity, C75 alters central and peripheral gene expression, which may lead to the profound reduction of adipose tissue in DIO mice.
Materials and Methods
DIO and lean mouse models
All animal experimentation was performed in accordance with guidelines on animal care and use as established by The Johns Hopkins University School of Medicine institutional animal care and use committee. Twelve-week-old, diet-induced obese C57BL6J male mice were obtained from The Jackson Laboratory (Bar Harbor, ME). DIO mice were fed a synthetic diet comprised of 60% calories from fat, 20% from carbohydrate, and 20% from protein (5.2 kcal/g) postweaning through the experimental procedures (D12492i, Research Diets, Inc., New Brunswick, NJ). Twelve-week-old C57BL6J male mice (The Jackson Laboratory), fed a diet of rodent chow comprised of 13% calories from fat, 58% from carbohydrate, and 29% from protein (4.1 kcal/g), were used for lean animal studies (Prolab RMH 2500, PMI Nutrition International, Inc., Brentwood, MO). Mice were maintained in a 12-h light, 12-h dark cycle at 25 C for 1 wk for acclimatization before treatment. C75 (FASgen, Inc., Baltimore, MD) was dissolved in RPMI 1640 (Invitrogen Life Technologies, Inc., Carlsbad, CA) and injected ip at 0900 h, approximately 3 h after lights-on, at the doses indicated.
Six DIO and lean mice were treated with C75 or vehicle every other day. An additional cohort of six mice was pair-fed to amounts consumed by the C75-treated animals in the prior 24 h. Body weight and food intake were measured daily. After completion of the treatment course, animals were euthanized by CO2 inhalation 4 h after the final dose of C75. Tissues were harvested immediately for RNA extraction.
Whole animal calorimetry
Oxygen consumption (VO2) and carbon dioxide production (VCO2) were measured in up to four mice at a time for each treatment group with indirect calorimetry (Oxymax Equal Flow System, Columbus Instruments, Columbus, OH). Measurements of VO2 (milliliters per kilogram per hour) and VCO2 (milliliters per kilogram per hour) were performed and recorded every 15 min. The respiratory exchange ratio (RER) was calculated using Oxymax software (version 5.9) and is defined as the ratio of VCO2 to VO2 (13). Calorimetry data are presented for the last 24 h of the 2-wk treatment, before collection of tissues for gene expression analysis.
RNA preparation and RT
Hypothalamus, liver, WAT, and muscle of DIO and lean mice were harvested and immediately frozen in liquid nitrogen. Total RNA was isolated with TRIzol reagent (Invitrogen Life Technologies, Inc.), according to the manufacturer’s instructions. RNA was quantified spectrophotometrically, and its quality was checked by agarose gel electrophoresis. RNA samples were treated with deoxyribonuclease I (amplification grade; Invitrogen Life Technologies, Inc.) to remove genomic DNA contamination. First-strand cDNA was synthesized from 1 μg total RNA in a 20-μl reaction volume using the ThermoScript RT-PCR System (Invitrogen Life Technologies, Inc.), according to the manufacturer’s instructions.
Real-time RT-PCR
Real-time quantitative RT-PCR was performed in a 25-μl reaction volume containing 500 nM of each primer [250 nM for AGRP, proopiomelanocortin (POMC), the muscle isoform of CPT1, PPAR, UCP2, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)], 12.5 μl 2x SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA), and 1 μl cDNA. Cycling conditions included an initial denaturation step at 95 C for 3 min, followed by 40 cycles of 95 C denaturation for 30 sec, 60 C (66 C for AGRP and POMC; 68 C for PPAR) annealing for 30 sec, and 72 C extension for 30 sec. Amplification and detection were performed on an iCycler iQ Real-Time PCR Detection System (Bio-Rad Laboratories). A negative control reaction in the absence of template was also performed for each primer pair. After completion of the cycling process, samples were subjected to a melting curve analysis to confirm the amplification specificity. Gene-specific primer pairs were designed using Primer3 software (www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi/). The sequences of the primer pairs are listed in Table 1.
TABLE 1. Primer pairs used in real-time RT-PCR
For each sample, the ratio between the relative amounts of target gene and GAPDH was calculated to compensate for variations in the quantity or quality of the starting mRNA as well as for differences in reverse transcriptase efficiency. The change in fluorescence of SYBR Green dye in every cycle was monitored, and the threshold cycle (CT) above background for each reaction was calculated. The fold change in target gene relative to the GAPDH endogenous control gene was determined by: fold change = 2–(CT), where CT = CT, target – CT, gapdh and (CT) = CT, treated – CT, control.
To validate the real-time RT-PCR method used in this study, we compared the expression of key genes in fatty acid metabolic pathways in liver, WAT, and skeletal muscle in DIO mice treated with vehicle as a control to determine whether our PCR expression levels were in keeping with reported values. The relative mRNA level was normalized to the highest expressing tissue as 1.0.
Acetyl-coenzyme A (acetyl-CoA) carboxylase (ACC) isoforms, FAS, and malonyl-CoA decarboxylase (MCD), enzymes involved in fatty acid synthesis, and glycerol-3-phosphate acyltransferases (GPAT), were present in 2-fold abundance in both liver and WAT. The liver isoform of CPT-1 (L-CPT-1) was nearly 200-fold more abundant in liver (1.0522) than in muscle (0.0059). Conversely, the muscle isoform of CPT-1 was about 150-fold more abundant in muscle (0.9991) than in liver (0.0081), both in keeping with published reports (14, 15). As previously reported, L-CPT-1 was the predominant isoform in mouse WAT (16). PPAR was most abundant in liver (1.0085 in liver, 0.0129 in WAT, and 0.0066 in muscle), with PPAR expressed predominantly in WAT (1.0102 in WAT, 0.0210 in liver, and 0.0081 in muscle) as reported previously (17, 18). UCP2 was the most abundant UCP in WAT and liver (1.0034 in WAT, 0.2037 in liver, and 0.0181 in muscle), with UCP3 predominating in muscle (1.0080 in muscle, 0.0007 in liver, and 0.4112 in WAT) (19, 20). Taken together, the patterns of expression of these genes in DIO mice were consistent with published reports, which validates this method for studying gene expression level in C75-treated animals.
Statistical analysis
All data are presented as the mean ± SE[SCAP];m of six independent measures/treatments. Data were analyzed by two-tailed unpaired t tests or one-way ANOVA where applicable, using PRISM 3.0 (GraphPad, San Diego, CA).
Results
C75 treatment of DIO and lean mice reduced body weight compared with pair-fed controls and reduced food consumption
To approximate a human model of obesity for the purpose of profiling changes in gene expression, we used an accepted DIO mouse model (13) and lean control animals treated with successive doses of C75 that reduced, but did not eliminate, food intake. Six DIO mice were treated initially with C75 at 10 mg/kg body weight, ip, on d 0 and 2, followed by maintenance doses of 5.0, 7.5, and 6.0 mg/kg every 48 h. Six lean mice were treated initially with C75 at 10 mg/kg, ip, on d 0 and 2, followed by maintenance doses of 7.5 mg/kg every 48 h. The goal of this C75 treatment protocol was to achieve a sustained and stable weight loss. Similar to prior acute (13) and chronic (1 month) (5) C75 treatment of DIO mice, the C75-treated DIO mice lost 17.3 ± 5.6% of their body weight compared with 4.6 ± 2.6% for the pair-fed animals (P = 0.015, by unpaired two-tailed t test), whereas vehicle controls lost 0.8 ± 4.0% (Fig. 1A). The C75-treated animals consumed less food per day on the average (Fig. 1B; 0.9 ± 0.2 g), compared with vehicle controls (2.3 ± 0.1 g; P < 0.0001, by unpaired one-tailed t test), demonstrating the anorexigenic effect of C75.
FIG. 1. Effects of C75 on body weight and food consumption in DIO and lean mice during a 2-wk treatment. Six DIO mice were treated initially with C75 at 10 mg/kg, ip, on d 0 and 2. Subsequent doses were administered as follows: 5.0 mg/kg on d 4; 7.5 mg/kg on d 6, 8, and 10; and 6.0 mg/kg on d 12. Six lean mice were treated initially with C75 at 10 mg/kg, ip, on d 0 and 2, followed by maintenance doses of 7.5 mg/kg on d 4, 6, 8, 10, and 12. The first two higher doses were used to obtain an initial big loss of body weight, and the subsequent five lower doses were used to maintain the initial body weight loss during the rest period in the 2-wk treatment procedure. The goal of this C75 treatment protocol was to achieve a sustained and stable weight loss. A, Body weights of vehicle control (black line), C75-treated (red line) and pair-fed (blue line) DIO mice were measured daily. C75 treatment caused a 17.3% reduction in body weight compared with 4.6% in the pair-fed group, whereas vehicle control animals lost less than 1% of their body weight. B, Food consumption of vehicle control (black line) and C75-treated (red line) DIO mice was measured daily. C75 treatment reduced average daily food consumption (0.8 g/d) compared with that of vehicle controls (2.3 g/d). C, Body weights of vehicle control (black line), C75-treated (red line), and pair-fed (blue line) lean mice were measured daily. In contrast to DIO mice, C75-treated lean mice lost 7.5% compared with 3.5% for the pair-fed group, whereas vehicle control mice gained 1.4%. D, Food consumption of vehicle control (black line) and C75-treated (red line) lean mice was measured daily. C75 treatment reduced food consumption (1.74 g/d) compared with vehicle controls (2.0 g/d). Data are expressed as the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
In lean C57BL6J male mice maintained on standard laboratory chow after weaning, C75 treatment resulted in a 7.5 ± 5.7% loss of body weight compared with a 3.5 ± 3.7% weight loss in the pair-fed group (P = 0.0024, by unpaired two-tailed t test), whereas vehicle controls gained 1.4 ± 1.6% (Fig. 1C). Control lean mice ate a daily average of 4.0 ± 0.1 g, whereas C75 treatment reduced food intake to 3.5 ± 0.3 g (P = 0.03, by unpaired one-tailed t test; Fig. 1D).
C75 treatment caused a persistent increase in fatty acid oxidation in DIO mice
DIO and lean mice treated with a single dose of C75 have been shown to increase fatty acid oxidation (13). In this study we monitored DIO and lean mice for the final 24 h after 2 wk of C75 treatment in the calorimeter to compare the in vivo metabolism with the gene expression profiling. In C75-treated DIO mice, VO2 averaged 4064 ± 48 ml/kg·h compared with 2725 ± 36 ml/kg·h for the pair-fed group, which represented an overall increase of 49% (Fig. 2A; P < 0.0001, by unpaired two-tailed t test). The RER was lower for C75-treated mice (0.84) than that for pair-fed mice (0.88; Fig. 2B; P < 0.0001, by unpaired two-tailed t test), indicating increased oxidation of fatty acids by the C75-treated animals. Taken together, these data indicate that 2-wk C75 treatment increased energy expenditure as fatty acid oxidation. Moreover, C75 maintained the ability to reduce food intake and increase energy expenditure throughout the 2-wk treatment regimen.
FIG. 2. Effect of C75 on energy expenditure in DIO and lean mice during the final 24 h of the 2-wk treatment. A, C75-treated mice maintained an average VO2 of 4064 ml/kg·h (red line) compared with 2725 ml/kg·h (black line) for the pair-fed group (P < 0.0001, by unpaired two-tailed t test). B, In contrast, the RER was lower for the C75-treated DIO mice (0.84; red line) compared with the pair-fed group (black line; 0.88; P < 0.0001, by unpaired two-tailed t test), indicating fatty acid oxidation. C, The average VO2 was essentially the same in both C75-treated lean animals (red line) and pair-fed controls (black line). D, In contrast, the RER was lower for the C75-treated lean mice 1.00 (red line) compared with 1.07 (black line) for the pair-fed group (P < 0.0001, by unpaired two-tailed t test). Error barsrepresent the SEM.
The findings in the C75-treated lean mice were similar to the effect of an acute single dose C75 treatment. There was no significant increase in VO2 in C75-treated lean animals compared with pair-fed controls (Fig. 2C). There was, however, a statistically significant decrease in RER with C75 treatment of 1.00 ± 0.01 compared with the pair-fed value of 1.07 ± 0.008 (Fig. 2D; P < 0.0001, by unpaired two-tailed t test). These data indicate no increase in energy expenditure, but a slight increase in oxidation of fatty acids, which could reflect the reduced food intake in the C75-treated lean animals. We summarize body weight, food consumption, VO2, and RER data in Table 2.
TABLE 2. Effect of C75 on body weight, food consumption, and energy expenditure
Gene expression alterations common to C75-treated and pair-fed DIO mice
In DIO mice, the expression of a series of genes changed coordinately in both C75-treated and pair-fed animals, consistent with the reduction of food intake common to both groups. In the liver (Fig. 3A), genes involved in fatty acid synthesis, FAS and ACC?, were down-regulated, whereas those responsible for fatty acid oxidation, L-CPT-1 and acyl-CoA oxidase (ACO) were nearly 2-fold up-regulated. Similarly in WAT (Fig. 3B), the fatty acid synthesis pathway genes, FAS and ACC, were also down-regulated. The expression of UCP3, the UCP selectively expressed in muscle, was increased by about 50% (Fig. 3C). The down-regulation of fatty acid synthesis genes in liver and WAT is expected with reduced food intake, along with up-regulation of fatty acid oxidation genes in the liver. The increased UCP3 in skeletal muscle has been seen in the setting of reduced food intake and increased fatty acid oxidation (21).
FIG. 3. Gene expression changes common to C75-treated and pair-fed DIO mice. Six mice were treated with vehicle () or C75 ( ) every other day. An additional cohort of six mice ( ) was pair-fed to the amounts consumed by the C75-treated animals. After 2 wk of treatment, tissues were harvested 4 h following the final dose. Total RNA (0.1 μg) was analyzed by real-time RT-PCR. A, The expression levels of ACC?, FAS, L-CPT1, and ACO were similar in the liver of C75-treated and pair-fed DIO mice. B, The expression levels of ACC and FAS were similar in the WAT of C75-treated and pair-fed DIO mice. C, UCP3 message expression was also similar in the muscle of C75-treated and pair-fed DIO mice. Data are expressed as the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Gene expression changes restricted to C75-treated DIO mice
Most of the C75-associated changes in the panel of genes whose expression we examined occurred in WAT (Fig. 4A). Again, the pattern of gene expression was consistent with up-regulation of fatty acid oxidation. Because ACC? is localized to the outer mitochondrial membrane (22), reduced ACC? expression would lead to lower ambient levels of malonyl-CoA in the vicinity of CPT-1, favoring increased fatty acid oxidation. Up-regulation of L-CPT-1 also promotes fatty acid oxidation. Although reduced MCD expression would not favor fatty acid oxidation per se, it did not alter the increased fatty acid oxidation noted in vivo.
FIG. 4. Gene expression changes restricted to C75-treated DIO mice. Six mice were treated with vehicle () or C75 ( ) every other day. An additional cohort of six mice ( ) was pair-fed to the amounts consumed by the C75-treated animals. After 2-wk treatment, tissues were harvested 4 h following the final dose. Total RNA (0.1 μg) was analyzed by real-time RT-PCR. A, The expression levels of ACC?, MCD, L-CPT1, PPAR, UCP2, and GPAT in WAT were examined in C75-treated DIO mice and were found to differ from those in control and pair-fed animals. B, The expression of PPAR and UCP2 was examined in liver of C75-treated DIO mice and was found to be different from that in pair-fed DIO mice. C, UCP2 expression was measured in C75-treated DIO mice in muscle and was increased compared with that in control and pair-fed mice. Data are expressed as the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
The genes responsible for promoting triglyceride synthesis and storage were also significantly down-regulated. Both PPAR and GPAT were down-regulated in WAT. In addition to promoting adipogenesis (17, 23), PPAR is thought to enhance postprandial triglyceride storage in adipocytes (24). GPAT, in particular the mitochondrial isoform, is the initial enzymatic step in the synthesis of triglycerides (25). Reduced expression of PPAR and GPAT would tend to reduce triglyceride accumulation in adipocytes. In addition, UCP2, also expressed in adipocytes, was 4-fold elevated compared with that in controls and 2-fold increased compared with that in pair-fed animals. Thus, in the adipocyte, C75 channels fatty acids away from storage by down-regulating PPAR and GPAT while enhancing fatty acid oxidation and mitochondrial uncoupling. The expression of this phenotype in the setting of reduced food consumption is responsible for the rapid loss of adipose tissue mass in C75-treated DIO mice.
In liver and muscle from DIO mice, C75 dramatically increased UCP2 expression, also accounting for the increased mitochondrial uncoupling (Fig. 4, B and C). Surprisingly, PPAR, which is responsible for the up-regulation of genes involved in fatty acid oxidation, was down-regulated by C75. This suggests that the increased expression of enzymes responsible for hepatic fatty acid oxidation seen with C75 is mediated via a mechanism distinct from PPAR.
Gene expression changes restricted to C75-treated lean mice
As in the DIO mice, most of the C75-associated changes in gene expression occurred in WAT (Fig. 5A). Genes responsible for fatty acid synthesis, FAS and ACC, were significantly reduced compared with those in pair-fed controls. PPAR expression was down-regulated 3-fold compared with that in pair-fed animals. GPAT expression was also reduced compared with that in both vehicle controls and pair-fed groups.
FIG. 5. Gene expression changes restricted to C75-treated lean mice. Six mice were treated with vehicle () or C75 ( ) every other day. An additional cohort of six mice ( ) was pair-fed to the amounts consumed by the C75-treated animals. After 2-wk treatment, tissues were harvested 4 h following the final dose. Total RNA (0.1 μg) was analyzed by real-time RT-PCR. A, The expression levels of message for ACC, FAS, PPAR, and GPAT were measured in C75-treated lean mice in WAT. B, The expression levels of PPAR and GPAT in liver were measured in C75-treated lean mice. In both tissues, changes unique to C75 treatment were demonstrated. Data are expressed as the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
In the liver of C75-treated mice (Fig. 5B), PPAR expression was also reduced nearly 2-fold compared with that in pair-fed mice. GPAT expression in C75-treated mice was similar to that in controls, but was reduced nearly 2-fold compared with that in the pair-fed group. Taken together, these data show that in lean animals, C75 did not enhance the expression of genes responsible for fatty acid oxidation. Instead, the combined reduction of de novo fatty acid synthesis and GPAT inhibition would probably lead to a net reduction of fatty acids available for triglyceride synthesis.
Alterations in hypothalamic neuropeptide expression in C75-treated DIO and lean mice
Similar to our studies of ob/ob and lean mice acutely treated with C75 (6), there was a reduction of NPY expression after 2 wk of C75 administration compared with that in pair-fed controls (Fig. 6A). In addition, AGRP expression was comparably reduced. Expression of the anorexigenic neuropeptides, POMC and cocaine-amphetamine-related transcript (CART), were increased compared with that in pair-fed animals, with no significant changes in melanin-concentrating hormone (MCH) expression. This hypothalamic profile of reduced orexigenic neuropeptide/increased anorexigenic neuropeptide expression is consistent with the significantly reduced food intake during the 2-wk treatment. In contrast, the hypothalamic profile of the lean mice (Fig. 6B) was notable for no changes in all three orexigenic neuropeptides, with significantly decreased expression of POMC and CART. This is in keeping with the similar food intake in C75-treated and control mice by the end of the 2-wk treatment.
FIG. 6. Effect of C75 on hypothalamic neuropeptide expression in DIO and lean mice. Six mice were treated with vehicle () or C75 ( ) every other day. An additional cohort of six mice ( ) was pair-fed to the amounts consumed by the C75-treated animals. After 2 wk of treatment, tissues were harvested 4 h following the final dose. Total hypothalamic RNA (0.1 μg) was analyzed by real-time RT-PCR. Hypothalamic neuropeptide expression was measured in DIO (A) and lean (B) mice. Data are expressed as the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Discussion
In the present study we quantified hypothalamic neuropeptide expression along with the expression of key genes involved in fatty acid metabolism in peripheral tissues after 2 wk of C75 treatment in DIO and lean mice using real-time RT-PCR. Similar to previously published studies (5, 13), we confirmed that C75 treatment significantly curtailed food intake and increased fatty acid oxidation, leading to dramatic weight loss in DIO mice. In lean mice, significant weight reduction occurred compared with pair-fed controls; however, indirect calorimetry failed to document a significant increase in energy expenditure as fatty acid oxidation during the final 24 h of the study. Although this finding does not rule out increased fatty acid oxidation in the lean animals during C75 administration, clearly, fatty acid oxidation was dramatically increased in DIO mice compared with that in the lean group.
Both C75 treatment and pair-feeding dramatically impacted fatty acid metabolism gene expression in DIO mice. A set of genes showed expression changes common to both the C75-treated and pair-fed groups, consistent with a response to reduced food consumption. This group included dramatic reductions in the lipogenic genes, ACC? and FAS, in both liver and WAT. ACC carboxylates acetyl-CoA to produce malonyl-CoA, the primary substrate for FAS. In liver, the expression of L-CTP-1 and ACO was increased along with that of UCP3 in muscle, consistent with increased fatty acid oxidation. CPT-1 esterifies long-chain acyl-CoAs to carnitine, thus allowing their passage into the mitochondrion for fatty acid oxidation (11, 12); ACO is the rate-limiting enzyme of peroxisomal fatty acid oxidation (26). Although the primary function of UCP3 is unclear, it is up-regulated in muscle as a response to increased fatty acid oxidation (27). Collectively, these findings are consistent with a normal physiological response to food deprivation consisting of decreased lipogenesis and increased fatty acid oxidation.
There were a number of genes whose expression changes were restricted to the C75-treated DIO group, and these most commonly occurred in WAT. Many of these gene expression changes favored oxidation of fatty acid over its incorporation into structural lipids or triglyceride. ACC? was dramatically down-regulated by C75 along with GPAT, whereas L-CPT-1 expression was increased. By reducing ACC? expression, C75 may reduce malonyl-CoA production in the vicinity of L-CPT-1, thus increasing L-CPT-1 activity while concomitantly increasing L-CPT-1 expression. These changes would establish a permissive state for the entrance of fatty acid into the mitochondria for oxidation. GPAT is the initial committed step for fatty acid incorporation into structural lipids or triglycerides (25). C75 inhibition of GPAT expression would further promote fatty acid oxidation by routing fatty acids away from storage and toward transport by the CPT-1 system for oxidation. C75 thus promotes both the shunting of fatty acid to oxidation and the entry of fatty acid into the mitochondria in WAT.
C75 also increased the expression of UCP2 in WAT, liver, and muscle. In addition to promoting uncoupling of mitochondrial oxidative phosphorylation, UCP2 has a role in limiting free radical formation during fatty acid oxidation (28). Through its mitochondrial uncoupling, increased UCP2 expression could allow for increased fatty acid oxidation without the necessity of producing excess ATP.
C75 also affected the expression of both PPAR and PPAR. The PPARs are a group of three nuclear receptor isoforms, PPAR, PPAR, and PPAR, encoded by different genes that regulate a variety of functions related to energy metabolism (29). PPAR is mainly expressed in liver, whereas PPAR is preferentially expressed in WAT. PPAR has been shown to play a critical role in the regulation of cellular uptake, activation, and ?-oxidation of fatty acid (29). Activation of PPAR directly up-regulates the transcription of both types of CPT-1 as well as ACO (30, 31, 32, 33). PPAR serves as a key regulator of adipocyte differentiation and lipid storage (17, 34). In DIO mice, C75 reduced the expression of PPAR in WAT, further promoting the reduction of lipid storage. In the liver, C75 reduced the expression of PPAR despite the increased expression of both L-CPT-1 and ACO. These data suggest that the pattern of gene expression favoring fatty acid oxidation was not due to increased PPAR activity, but was caused by an as yet undetermined mechanism. Accomplishing increased fatty acid oxidation without enhancing PPAR expression could have a particular advantage to cardiac muscle. Increased PPAR expression increases fatty acid transport beyond the capacity of increased fatty acid oxidation, leading to fatty acid deposition in cardiac muscle. This is thought to be the mechanism responsible for diabetic cardiomyopathy (35). Real-time RT-PCR measurements of cardiac muscle after 1 month of C75 treatment failed to show any increase in PPAR expression (data not shown).
In the lean mice, there were no genes with expression changes common to both the C75 and pair-fed groups. A number of genes, however, showed expression changes restricted to C75 treatment. In response to C75, lipogenesis was reduced in WAT, as evidenced by decreased expression of ACC and FAS. Although PPAR expression was reduced, its significance is unknown, because it is not highly expressed in WAT. GPAT expression was reduced in WAT and also in liver along with PPAR. Taken together, these changes indicate a C75-driven reduction in fatty acid synthesis, with a potential for increased fatty acid oxidation due to the reduction of GPAT expression.
Differences between the effects of C75 on DIO and lean mice might be due to different peripheral metabolisms. In DIO mice, C75 increased energy expenditure as fatty acid oxidation manifested as increased VO2 and reduced RER compared with those in pair-fed animals. These data indicate that energy expenditure in DIO mice was mainly from fatty acid oxidation. In lean mice, the diet was high in carbohydrates, consistent with the high RER levels. Although there was no significant increase in energy expenditure with C75, there was a modest reduction in RER compared with pair-fed animals, indicating increased oxidation of fatty acids over glucose. This is in keeping with the gene expression analysis showing that genes involved in fatty acid oxidation were only induced by C75 in DIO mice, not in lean mice. The combination of altered gene expression and the high fat diet in DIO mice is probably responsible for the metabolic differences between lean and DIO mice.
Studies have demonstrated fundamental differences in hypothalamic neuropeptide responses between DIO and lean rodents (36, 37, 38, 39, 40). Consistent with these models, C75 treatment had qualitatively different effects on hypothalamic neuropeptide expression in DIO and lean mice in this study. C75 dramatically inhibited orexigenic neuropeptides (NPY and AGRP) expression and induced anorexigenic neuropeptides (POMC and CART) expression in DIO mice. In lean mice, C75 decreased the expression of anorexigenic neuropeptides (POMC and CART) without changes in the expression of orexigenic neuropeptides (NPY and AGRP). Despite the difference in methodology and number of samples, the overall pattern of expression is similar to that in our prior study (5) and reflects the levels of food consumption. The DIO mice continued to exhibit reduced food intake during the entire duration of treatment, and they displayed a strongly anorexigenic neuropeptide profile. In contrast, by the conclusion of treatment, the lean mice were eating an amount nearly equivalent to controls, and they had a modestly orexigenic hypothalamic profile.
The results of our fatty acid metabolism gene expression analysis advance our understanding of the selectivity of C75 in reducing adipose tissue mass. Conceptually, the dramatic increase in fatty acid oxidation in C75-treated DIO mice would probably require more than competitive stimulation of CPT-1. The gene expression changes in WAT from C75-treated DIO mice increase the expression of enzymes responsible for both the transport of fatty acid into the mitochondria and the shunting of fatty acids away from anabolic pathways to oxidative catabolism. Moreover, the increase in UCP2 expression in liver, WAT, and muscle would allow for increased fatty acid oxidation by mitochondrial uncoupling without requiring superphysiological production of ATP and excessive free radical generation. Interestingly, additional studies have shown that the weight loss effect of C75 treatment persists for nearly 2 wk beyond cessation of therapy (data not shown), also supporting the importance of these gene expression changes for C75 weight maintenance. The exploration of the mechanism of action of C75 serves to further our understanding of the biological consequences of fatty acid synthesis inhibition and fatty acid oxidation stimulation in vivo.
Acknowledgments
We thank members of the FAS Working Group for critical reading of this manuscript.
References
Gale SM, Castracane VD, Mantzoros CS 2004 Energy homeostasis, obesity and eating disorders: recent advances in endocrinology. J Nutr 134:295–298
Must A, Spadano J, Coakley EH, Field AE, Colditz G, Dietz WH 1999 The disease burden associated with overweight and obesity. JAMA 282:1523–1529
Kopelman P 2000 Obesity as a medical problem. Nature 404:635–643
Glazer G 2001 Long-term pharmacotherapy of obesity 2000: a review of efficacy and safety. Arch Intern Med 161:1814–1824
Thupari JN, Kim EK, Moran TH, Ronnett GV, Kuhajda FP 2004 Chronic C75 treatment of diet-induced obese mice increases fat oxidation and reduces food intake to reduce adipose mass. Am J Physiol 287:E97–E104
Loftus T, Jaworsky D, Frehywot G, Townsend CA, Ronnett GV, Lane MD, Kuhajda FP 2000 Reduced food intake and body weight in mice treated with fatty acid synthase inhibitors. Science 288:2379–2381
Kim EK, Miller I, Landree LE, Borisy-Rudin FF, Brown P, Tihan T, Townsend CA, Witters LA, Moran TH, Kuhajda FP, Ronnett GV 2002 Expression of FAS within hypothalamic neurons: a model for decreased food intake after C75 treatment. Am J Physiol 283:E867–E879
Kumar MV, Shimokawa T, Nagy TR, Lane MD 2002 Differential effects of a centrally acting fatty acid synthase inhibitor in lean and obese mice. Proc Natl Acad Sci USA 99:1921–1925
Shimokawa T, Kumar MV, Lane MD 2002 Effect of a fatty acid synthase inhibitor on food intake and expression of hypothalamic neuropeptides. Proc Natl Acad Sci USA 99:66–71
Kim EK, Miller I, Aja S, Landree LE, Pinn M, McFadden J, Kuhajda FP, Moran TH, Ronnett GV 2004 C75, a fatty acid synthase inhibitor, reduces food intake via hypothalamic AMP-activated protein kinase. J Biol Chem 279:19970–19976
McGarry JD, Brown NF 1997 The mitochondrial carnitine palmitoyltransferase system from concept to molecular analysis. Eur J Biochem 244:1–14
Eaton S, Bartlett K, Quant PA 2001 Carnitine palmitoyl transferase I and the control of ?-oxidation in heart mitochondria. Biochem Biophys Res Commun 285:537–539
Thupari JN, Landree LE, Ronnett GV, Kuhajda FP 2002 C75 increases peripheral energy utilization and fatty acid oxidation in diet-induced obesity. Proc Natl Acad Sci USA 99:9498–9502
Woeltje KF, Esser V, Weis BC, Cox WF, Schroeder JG, Liao ST, Foster DW, McGarry JD 1990 Inter-tissue and inter-species characteristics of the mitochondrial carnitine palmitoyltransferase enzyme system. J Biol Chem 265:10714–10719
Weis BC, Esser V, Foster DW, McGarry JD 1994 Rat heart expresses two forms of mitochondrial carnitine palmitoyltransferase I. The minor component is identical to the liver enzyme. J Biol Chem 269:18712–18715
Esser V, Brown NF, Cowan AT, Foster DW, McGarry JD 1996 Expression of a cDNA isolated from rat brown adipose tissue and heart identifies the product as the muscle isoform of carnitine palmitoyltransferase I (M-CPT-I). J Biol Chem 271:6972–6977
Kersten S 2002 Peroxisome proliferator activated receptors and obesity. Eur J Pharmacol 440:223–234
Bocher V, Pineda-Torra I, Fruchart JC, Staels B 2002 PPARs: transcription factors controlling lipid and lipoprotein metabolism. Ann NY Acad Sci 967:7–18
Jezek P 2002 Possible physiological roles of mitochondrial uncoupling proteins: UCPn. Int J Biochem Cell Biol 34:1190–1206
Ledesma A, de Lacoba MG, Rial E 2002 The mitochondrial uncoupling proteins. Genome Biol 3:Reviews3015.1–3015.9
Cha SH, Hu Z, Lane MD 2004 Long-term effects of a fatty acid synthase inhibitor on obese mice: food intake, hypothalamic neuropeptides, and UCP3. Biochem Biophys Res Commun 317:301–308
Ha J, Lee JK, Kim KS, Witters LA, Kim KH 1996 Cloning of human acetyl-CoA carboxylase-? and its unique features. Proc Natl Acad Sci USA 93:11466–11470
Lemberger T, Desvergne B, Wahli W 1996 Peroxisome proliferator-activated receptors: a nuclear receptor signaling pathway in lipid physiology. Annu Rev Cell Dev Biol 12:335–363
Laplante M, Sell H, MacNaul KL, Richard D, Berger JP, Deshaies Y 2003 PPAR- activation mediates adipose depot-specific effects on gene expression and lipoprotein lipase activity: mechanisms for modulation of postprandial lipemia and differential adipose accretion. Diabetes 52:291–299
Dircks LK, Sul HS 1997 Mammalian mitochondrial glycerol-3-phosphate acyltransferase. Biochim Biophys Acta 1348:17–26
Nohammer C, El-Shabrawi Y, Schauer S, Hiden M, Berger J, Forss-Petter S, Winter E, Eferl R, Zechner R, Hoefler G 2000 cDNA cloning and analysis of tissue-specific expression of mouse peroxisomal straight-chain acyl-CoA oxidase. Eur J Biochem 267:1254–1260
Himms-Hagen J, Harper ME 2001 Physiological role of UCP3 may be export of fatty acids from mitochondria when fatty acid oxidation predominates: an hypothesis. Exp Biol Med 226:78–84
Echtay KS, Roussel D, St-Pierre J, Jekabsons MB, Cadenas S, Stuart JA, Harper JA, Roebuck SJ, Morrison A, Pickering S, Clapham JC, Brand MD 2002 Superoxide activates mitochondrial uncoupling proteins. Nature 415:96–99
Berger J, Moller D 2002 The mechanisms of action of PPARs. Annu Rev Med 53:409–435
Brady PS, Marine KA, Brady LJ, Ramsay RR 1989 Co-ordinate induction of hepatic mitochondrial and peroxisomal carnitine acyltransferase synthesis by diet and drugs. Biochem J 260:93–100
Dreyer C, Krey G, Keller H, Givel F, Helftenbein G, Wahli W 1992 Control of the peroxisomal ?-oxidation pathway by a novel family of nuclear hormone receptors. Cell 68:879–887
Tugwood JD, Issemann I, Anderson RG, Bundell KR, McPheat WL, Green S 1992 The mouse peroxisome proliferator activated receptor recognizes a response element in the 5' flanking sequence of the rat acyl CoA oxidase gene. EMBO J 11:433–439
Mascaro C, Acosta E, Ortiz JA, Marrero PF, Hegardt FG, Haro D 1998 Control of human muscle-type carnitine palmitoyltransferase I gene transcription by peroxisome proliferator-activated receptor. J Biol Chem 273:8560–8563
Gerhold DL, Liu F, Jiang G, Li Z, Xu J, Lu M, Sachs JR, Bagchi A, Fridman A, Holder DJ, Doebber TW, Berger J, Elbrecht A, Moller DE, Zhang BB 2002 Gene expression profile of adipocyte differentiation and its regulation by peroxisome proliferator-activated receptor- agonists. Endocrinology 143:2106–2118
Finck BN, Lehman JJ, Leone TC, Welch MJ, Bennett MJ, Kovacs A, Han X, Gross RW, Kozak R, Lopaschuk GD, Kelly DP 2002 The cardiac phenotype induced by PPAR overexpression mimics that caused by diabetes mellitus. J Clin Invest 109:121–130
Bergen HT, Mizuno T, Taylor J, Mobbs CV 1999 Resistance to diet-induced obesity is associated with increased proopiomelanocortin mRNA and decreased neuropeptide Y mRNA in the hypothalamus. Brain Res 851:198–203
Levin BE, Dunn-Meynell AA 2000 Sibutramine alters the central mechanisms regulating the defended body weight in diet-induced obese rats. Am J Physiol 279:R2222–R2228
Ziotopoulou M, Mantzoros CS, Hileman SM, Flier JS 2000 Differential expression of hypothalamic neuropeptides in the early phase of diet-induced obesity in mice. Am J Physiol 279:E838–E845
Wang H, Storlien LH, Huang X 2002 Effects of dietary fat types on body fatness, leptin, and ARC leptin receptor, NPY, and AgRP mRNA expression. Am J Physiol 282:E1352–E1359
Rohner-Jeanrenaud F, Craft LS, Bridwell J, Suter TM, Tinsley FC, Smiley DL, Burkhart DR, Statnick MA, Heiman ML, Ravussin E, Caro JF 2002 Chronic central infusion of cocaine- and amphetamine-regulated transcript (CART 55–102): effects on body weight homeostasis in lean and high-fat-fed obese rats. Int J Obes Relat Metab Disord 26:143–149(Yajun Tu, Jagan N. Thupar)
Address all correspondence and requests for reprints to: Dr. Gabriele V. Ronnett, Department of Neuroscience, 1006B Preclinical Teaching Building, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205. E-mail: gronnett@jhmi.edu.
Abstract
C75, a synthetic inhibitor of fatty acid synthase (FAS), causes anorexia and profound weight loss in lean and genetically obese mice. C75 also acts as a stimulator of carnitine palmitoyltransferase-1 to induce fatty acid oxidation. To approximate human obesity, we used a 2-wk C75 treatment model for diet-induced obese (DIO) mice to investigate the central and peripheral effects of C75 on gene expression. C75 treatment decreased food intake, increased energy expenditure, and reduced body weight more effectively in DIO than in lean mice. Analysis of the gene expression changes in hypothalamus demonstrated that the reduced food intake in C75-treated DIO mice might be mediated by inhibition of orexigenic neuropeptide expression and induction of anorexigenic neuropeptide expression. Gene expression changes in peripheral tissues indicated that C75 increased energy expenditure by the induction of genes involved in fatty acid oxidation. C75 also inhibited the expression of genes in peripheral tissues responsible for fatty acid synthesis and accumulation. The patterns of the changes in central and peripheral gene expression that occur with C75 treatment provide mechanisms to explain the reduced food intake and increased energy expenditure observed with C75.
Introduction
OBESITY HAS NOW surpassed tobacco as the leading cause of preventable mortality in the United States (1). Resulting from a chronic imbalance between energy intake and energy expenditure, obesity is frequently associated with other diseases, such as diabetes and hypertension (2, 3). Although appropriate diet and exercise may lead to a reduction in adipose mass, many patients eventually regain the weight (4). Thus, pharmacological treatment may be required for the control of obesity in many patients.
C75, a compound designed as an inhibitor of fatty acid synthase (FAS), causes reduced food consumption and increased fatty acid oxidation in diet-induced obese (DIO) mice, leading to profound loss of adipose tissue (5). Studies performed by us and others have shown that the anorexigenic effects of C75 are mediated through alterations of hypothalamic neuropeptide expression (5, 6, 7, 8, 9). C75 reduces neuropeptide Y (NPY) and agouti-related protein (AGRP) expression in lean mice in acute treatment experiments (5, 6, 7). We and others have also shown that C75 affects hypothalamic neuropeptide expression in lean and obese (ob/ob) mice in acute treatment experiments (9) and in lean, obese (ob/ob), and DIO mice in multiple-day treatment experiments (5, 8). Moreover, the central effect of C75 on the expression of hypothalamic neuropeptides is probably mediated through C75 modulation of neuronal ATP levels and ensuing alterations in AMP-activated protein kinase phosphorylation and activity (10). The increased fatty acid oxidation noted both in vitro and in vivo has been shown to be due at least in part to direct C75 stimulation of carnitine palmitoyltransferase-1 (CPT-1), the pace-setting enzyme of mitochondrial fatty acid oxidation (11, 12).
In addition to affecting hypothalamic neuropeptide expression, C75 could potentially alter the expression of genes involved in fatty acid metabolism due to its direct effects on fatty acid synthesis and oxidation. In this study we used a 2-wk chronic C75 treatment model suitable for both DIO and lean mice and examined the effects of C75 on body weight, food intake, and energy expenditure. We also quantified the expression of key genes involved in fatty acid metabolism in liver, white adipose tissue (WAT) and skeletal muscle along with hypothalamic neuropeptide expression.
Our results showed that the 2-wk C75 treatment was more efficacious in DIO mice than in lean mice, as evidenced by the increased weight loss, decreased food intake, and increased energy expenditure in DIO mice. In the hypothalamus of DIO mice, C75 treatment caused a marked anorexigenic neuropeptide profile, with reduction of orexigenic neuropeptide expression and increased anorexigenic neuropeptide expression. In WAT obtained from DIO mice, C75 treatment altered the expression of genes involved in fatty acid metabolism to favor fatty acid oxidation, without increasing the expression of peroxisome proliferator-activated receptor(PPAR). Uncoupling protein 2 (UCP2) expression was also increased in liver, WAT, and skeletal muscle of C75-treated DIO mice. Thus, in addition to direct effects on FAS and CPT-1 enzyme activity, C75 alters central and peripheral gene expression, which may lead to the profound reduction of adipose tissue in DIO mice.
Materials and Methods
DIO and lean mouse models
All animal experimentation was performed in accordance with guidelines on animal care and use as established by The Johns Hopkins University School of Medicine institutional animal care and use committee. Twelve-week-old, diet-induced obese C57BL6J male mice were obtained from The Jackson Laboratory (Bar Harbor, ME). DIO mice were fed a synthetic diet comprised of 60% calories from fat, 20% from carbohydrate, and 20% from protein (5.2 kcal/g) postweaning through the experimental procedures (D12492i, Research Diets, Inc., New Brunswick, NJ). Twelve-week-old C57BL6J male mice (The Jackson Laboratory), fed a diet of rodent chow comprised of 13% calories from fat, 58% from carbohydrate, and 29% from protein (4.1 kcal/g), were used for lean animal studies (Prolab RMH 2500, PMI Nutrition International, Inc., Brentwood, MO). Mice were maintained in a 12-h light, 12-h dark cycle at 25 C for 1 wk for acclimatization before treatment. C75 (FASgen, Inc., Baltimore, MD) was dissolved in RPMI 1640 (Invitrogen Life Technologies, Inc., Carlsbad, CA) and injected ip at 0900 h, approximately 3 h after lights-on, at the doses indicated.
Six DIO and lean mice were treated with C75 or vehicle every other day. An additional cohort of six mice was pair-fed to amounts consumed by the C75-treated animals in the prior 24 h. Body weight and food intake were measured daily. After completion of the treatment course, animals were euthanized by CO2 inhalation 4 h after the final dose of C75. Tissues were harvested immediately for RNA extraction.
Whole animal calorimetry
Oxygen consumption (VO2) and carbon dioxide production (VCO2) were measured in up to four mice at a time for each treatment group with indirect calorimetry (Oxymax Equal Flow System, Columbus Instruments, Columbus, OH). Measurements of VO2 (milliliters per kilogram per hour) and VCO2 (milliliters per kilogram per hour) were performed and recorded every 15 min. The respiratory exchange ratio (RER) was calculated using Oxymax software (version 5.9) and is defined as the ratio of VCO2 to VO2 (13). Calorimetry data are presented for the last 24 h of the 2-wk treatment, before collection of tissues for gene expression analysis.
RNA preparation and RT
Hypothalamus, liver, WAT, and muscle of DIO and lean mice were harvested and immediately frozen in liquid nitrogen. Total RNA was isolated with TRIzol reagent (Invitrogen Life Technologies, Inc.), according to the manufacturer’s instructions. RNA was quantified spectrophotometrically, and its quality was checked by agarose gel electrophoresis. RNA samples were treated with deoxyribonuclease I (amplification grade; Invitrogen Life Technologies, Inc.) to remove genomic DNA contamination. First-strand cDNA was synthesized from 1 μg total RNA in a 20-μl reaction volume using the ThermoScript RT-PCR System (Invitrogen Life Technologies, Inc.), according to the manufacturer’s instructions.
Real-time RT-PCR
Real-time quantitative RT-PCR was performed in a 25-μl reaction volume containing 500 nM of each primer [250 nM for AGRP, proopiomelanocortin (POMC), the muscle isoform of CPT1, PPAR, UCP2, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)], 12.5 μl 2x SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA), and 1 μl cDNA. Cycling conditions included an initial denaturation step at 95 C for 3 min, followed by 40 cycles of 95 C denaturation for 30 sec, 60 C (66 C for AGRP and POMC; 68 C for PPAR) annealing for 30 sec, and 72 C extension for 30 sec. Amplification and detection were performed on an iCycler iQ Real-Time PCR Detection System (Bio-Rad Laboratories). A negative control reaction in the absence of template was also performed for each primer pair. After completion of the cycling process, samples were subjected to a melting curve analysis to confirm the amplification specificity. Gene-specific primer pairs were designed using Primer3 software (www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi/). The sequences of the primer pairs are listed in Table 1.
TABLE 1. Primer pairs used in real-time RT-PCR
For each sample, the ratio between the relative amounts of target gene and GAPDH was calculated to compensate for variations in the quantity or quality of the starting mRNA as well as for differences in reverse transcriptase efficiency. The change in fluorescence of SYBR Green dye in every cycle was monitored, and the threshold cycle (CT) above background for each reaction was calculated. The fold change in target gene relative to the GAPDH endogenous control gene was determined by: fold change = 2–(CT), where CT = CT, target – CT, gapdh and (CT) = CT, treated – CT, control.
To validate the real-time RT-PCR method used in this study, we compared the expression of key genes in fatty acid metabolic pathways in liver, WAT, and skeletal muscle in DIO mice treated with vehicle as a control to determine whether our PCR expression levels were in keeping with reported values. The relative mRNA level was normalized to the highest expressing tissue as 1.0.
Acetyl-coenzyme A (acetyl-CoA) carboxylase (ACC) isoforms, FAS, and malonyl-CoA decarboxylase (MCD), enzymes involved in fatty acid synthesis, and glycerol-3-phosphate acyltransferases (GPAT), were present in 2-fold abundance in both liver and WAT. The liver isoform of CPT-1 (L-CPT-1) was nearly 200-fold more abundant in liver (1.0522) than in muscle (0.0059). Conversely, the muscle isoform of CPT-1 was about 150-fold more abundant in muscle (0.9991) than in liver (0.0081), both in keeping with published reports (14, 15). As previously reported, L-CPT-1 was the predominant isoform in mouse WAT (16). PPAR was most abundant in liver (1.0085 in liver, 0.0129 in WAT, and 0.0066 in muscle), with PPAR expressed predominantly in WAT (1.0102 in WAT, 0.0210 in liver, and 0.0081 in muscle) as reported previously (17, 18). UCP2 was the most abundant UCP in WAT and liver (1.0034 in WAT, 0.2037 in liver, and 0.0181 in muscle), with UCP3 predominating in muscle (1.0080 in muscle, 0.0007 in liver, and 0.4112 in WAT) (19, 20). Taken together, the patterns of expression of these genes in DIO mice were consistent with published reports, which validates this method for studying gene expression level in C75-treated animals.
Statistical analysis
All data are presented as the mean ± SE[SCAP];m of six independent measures/treatments. Data were analyzed by two-tailed unpaired t tests or one-way ANOVA where applicable, using PRISM 3.0 (GraphPad, San Diego, CA).
Results
C75 treatment of DIO and lean mice reduced body weight compared with pair-fed controls and reduced food consumption
To approximate a human model of obesity for the purpose of profiling changes in gene expression, we used an accepted DIO mouse model (13) and lean control animals treated with successive doses of C75 that reduced, but did not eliminate, food intake. Six DIO mice were treated initially with C75 at 10 mg/kg body weight, ip, on d 0 and 2, followed by maintenance doses of 5.0, 7.5, and 6.0 mg/kg every 48 h. Six lean mice were treated initially with C75 at 10 mg/kg, ip, on d 0 and 2, followed by maintenance doses of 7.5 mg/kg every 48 h. The goal of this C75 treatment protocol was to achieve a sustained and stable weight loss. Similar to prior acute (13) and chronic (1 month) (5) C75 treatment of DIO mice, the C75-treated DIO mice lost 17.3 ± 5.6% of their body weight compared with 4.6 ± 2.6% for the pair-fed animals (P = 0.015, by unpaired two-tailed t test), whereas vehicle controls lost 0.8 ± 4.0% (Fig. 1A). The C75-treated animals consumed less food per day on the average (Fig. 1B; 0.9 ± 0.2 g), compared with vehicle controls (2.3 ± 0.1 g; P < 0.0001, by unpaired one-tailed t test), demonstrating the anorexigenic effect of C75.
FIG. 1. Effects of C75 on body weight and food consumption in DIO and lean mice during a 2-wk treatment. Six DIO mice were treated initially with C75 at 10 mg/kg, ip, on d 0 and 2. Subsequent doses were administered as follows: 5.0 mg/kg on d 4; 7.5 mg/kg on d 6, 8, and 10; and 6.0 mg/kg on d 12. Six lean mice were treated initially with C75 at 10 mg/kg, ip, on d 0 and 2, followed by maintenance doses of 7.5 mg/kg on d 4, 6, 8, 10, and 12. The first two higher doses were used to obtain an initial big loss of body weight, and the subsequent five lower doses were used to maintain the initial body weight loss during the rest period in the 2-wk treatment procedure. The goal of this C75 treatment protocol was to achieve a sustained and stable weight loss. A, Body weights of vehicle control (black line), C75-treated (red line) and pair-fed (blue line) DIO mice were measured daily. C75 treatment caused a 17.3% reduction in body weight compared with 4.6% in the pair-fed group, whereas vehicle control animals lost less than 1% of their body weight. B, Food consumption of vehicle control (black line) and C75-treated (red line) DIO mice was measured daily. C75 treatment reduced average daily food consumption (0.8 g/d) compared with that of vehicle controls (2.3 g/d). C, Body weights of vehicle control (black line), C75-treated (red line), and pair-fed (blue line) lean mice were measured daily. In contrast to DIO mice, C75-treated lean mice lost 7.5% compared with 3.5% for the pair-fed group, whereas vehicle control mice gained 1.4%. D, Food consumption of vehicle control (black line) and C75-treated (red line) lean mice was measured daily. C75 treatment reduced food consumption (1.74 g/d) compared with vehicle controls (2.0 g/d). Data are expressed as the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
In lean C57BL6J male mice maintained on standard laboratory chow after weaning, C75 treatment resulted in a 7.5 ± 5.7% loss of body weight compared with a 3.5 ± 3.7% weight loss in the pair-fed group (P = 0.0024, by unpaired two-tailed t test), whereas vehicle controls gained 1.4 ± 1.6% (Fig. 1C). Control lean mice ate a daily average of 4.0 ± 0.1 g, whereas C75 treatment reduced food intake to 3.5 ± 0.3 g (P = 0.03, by unpaired one-tailed t test; Fig. 1D).
C75 treatment caused a persistent increase in fatty acid oxidation in DIO mice
DIO and lean mice treated with a single dose of C75 have been shown to increase fatty acid oxidation (13). In this study we monitored DIO and lean mice for the final 24 h after 2 wk of C75 treatment in the calorimeter to compare the in vivo metabolism with the gene expression profiling. In C75-treated DIO mice, VO2 averaged 4064 ± 48 ml/kg·h compared with 2725 ± 36 ml/kg·h for the pair-fed group, which represented an overall increase of 49% (Fig. 2A; P < 0.0001, by unpaired two-tailed t test). The RER was lower for C75-treated mice (0.84) than that for pair-fed mice (0.88; Fig. 2B; P < 0.0001, by unpaired two-tailed t test), indicating increased oxidation of fatty acids by the C75-treated animals. Taken together, these data indicate that 2-wk C75 treatment increased energy expenditure as fatty acid oxidation. Moreover, C75 maintained the ability to reduce food intake and increase energy expenditure throughout the 2-wk treatment regimen.
FIG. 2. Effect of C75 on energy expenditure in DIO and lean mice during the final 24 h of the 2-wk treatment. A, C75-treated mice maintained an average VO2 of 4064 ml/kg·h (red line) compared with 2725 ml/kg·h (black line) for the pair-fed group (P < 0.0001, by unpaired two-tailed t test). B, In contrast, the RER was lower for the C75-treated DIO mice (0.84; red line) compared with the pair-fed group (black line; 0.88; P < 0.0001, by unpaired two-tailed t test), indicating fatty acid oxidation. C, The average VO2 was essentially the same in both C75-treated lean animals (red line) and pair-fed controls (black line). D, In contrast, the RER was lower for the C75-treated lean mice 1.00 (red line) compared with 1.07 (black line) for the pair-fed group (P < 0.0001, by unpaired two-tailed t test). Error barsrepresent the SEM.
The findings in the C75-treated lean mice were similar to the effect of an acute single dose C75 treatment. There was no significant increase in VO2 in C75-treated lean animals compared with pair-fed controls (Fig. 2C). There was, however, a statistically significant decrease in RER with C75 treatment of 1.00 ± 0.01 compared with the pair-fed value of 1.07 ± 0.008 (Fig. 2D; P < 0.0001, by unpaired two-tailed t test). These data indicate no increase in energy expenditure, but a slight increase in oxidation of fatty acids, which could reflect the reduced food intake in the C75-treated lean animals. We summarize body weight, food consumption, VO2, and RER data in Table 2.
TABLE 2. Effect of C75 on body weight, food consumption, and energy expenditure
Gene expression alterations common to C75-treated and pair-fed DIO mice
In DIO mice, the expression of a series of genes changed coordinately in both C75-treated and pair-fed animals, consistent with the reduction of food intake common to both groups. In the liver (Fig. 3A), genes involved in fatty acid synthesis, FAS and ACC?, were down-regulated, whereas those responsible for fatty acid oxidation, L-CPT-1 and acyl-CoA oxidase (ACO) were nearly 2-fold up-regulated. Similarly in WAT (Fig. 3B), the fatty acid synthesis pathway genes, FAS and ACC, were also down-regulated. The expression of UCP3, the UCP selectively expressed in muscle, was increased by about 50% (Fig. 3C). The down-regulation of fatty acid synthesis genes in liver and WAT is expected with reduced food intake, along with up-regulation of fatty acid oxidation genes in the liver. The increased UCP3 in skeletal muscle has been seen in the setting of reduced food intake and increased fatty acid oxidation (21).
FIG. 3. Gene expression changes common to C75-treated and pair-fed DIO mice. Six mice were treated with vehicle () or C75 ( ) every other day. An additional cohort of six mice ( ) was pair-fed to the amounts consumed by the C75-treated animals. After 2 wk of treatment, tissues were harvested 4 h following the final dose. Total RNA (0.1 μg) was analyzed by real-time RT-PCR. A, The expression levels of ACC?, FAS, L-CPT1, and ACO were similar in the liver of C75-treated and pair-fed DIO mice. B, The expression levels of ACC and FAS were similar in the WAT of C75-treated and pair-fed DIO mice. C, UCP3 message expression was also similar in the muscle of C75-treated and pair-fed DIO mice. Data are expressed as the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Gene expression changes restricted to C75-treated DIO mice
Most of the C75-associated changes in the panel of genes whose expression we examined occurred in WAT (Fig. 4A). Again, the pattern of gene expression was consistent with up-regulation of fatty acid oxidation. Because ACC? is localized to the outer mitochondrial membrane (22), reduced ACC? expression would lead to lower ambient levels of malonyl-CoA in the vicinity of CPT-1, favoring increased fatty acid oxidation. Up-regulation of L-CPT-1 also promotes fatty acid oxidation. Although reduced MCD expression would not favor fatty acid oxidation per se, it did not alter the increased fatty acid oxidation noted in vivo.
FIG. 4. Gene expression changes restricted to C75-treated DIO mice. Six mice were treated with vehicle () or C75 ( ) every other day. An additional cohort of six mice ( ) was pair-fed to the amounts consumed by the C75-treated animals. After 2-wk treatment, tissues were harvested 4 h following the final dose. Total RNA (0.1 μg) was analyzed by real-time RT-PCR. A, The expression levels of ACC?, MCD, L-CPT1, PPAR, UCP2, and GPAT in WAT were examined in C75-treated DIO mice and were found to differ from those in control and pair-fed animals. B, The expression of PPAR and UCP2 was examined in liver of C75-treated DIO mice and was found to be different from that in pair-fed DIO mice. C, UCP2 expression was measured in C75-treated DIO mice in muscle and was increased compared with that in control and pair-fed mice. Data are expressed as the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
The genes responsible for promoting triglyceride synthesis and storage were also significantly down-regulated. Both PPAR and GPAT were down-regulated in WAT. In addition to promoting adipogenesis (17, 23), PPAR is thought to enhance postprandial triglyceride storage in adipocytes (24). GPAT, in particular the mitochondrial isoform, is the initial enzymatic step in the synthesis of triglycerides (25). Reduced expression of PPAR and GPAT would tend to reduce triglyceride accumulation in adipocytes. In addition, UCP2, also expressed in adipocytes, was 4-fold elevated compared with that in controls and 2-fold increased compared with that in pair-fed animals. Thus, in the adipocyte, C75 channels fatty acids away from storage by down-regulating PPAR and GPAT while enhancing fatty acid oxidation and mitochondrial uncoupling. The expression of this phenotype in the setting of reduced food consumption is responsible for the rapid loss of adipose tissue mass in C75-treated DIO mice.
In liver and muscle from DIO mice, C75 dramatically increased UCP2 expression, also accounting for the increased mitochondrial uncoupling (Fig. 4, B and C). Surprisingly, PPAR, which is responsible for the up-regulation of genes involved in fatty acid oxidation, was down-regulated by C75. This suggests that the increased expression of enzymes responsible for hepatic fatty acid oxidation seen with C75 is mediated via a mechanism distinct from PPAR.
Gene expression changes restricted to C75-treated lean mice
As in the DIO mice, most of the C75-associated changes in gene expression occurred in WAT (Fig. 5A). Genes responsible for fatty acid synthesis, FAS and ACC, were significantly reduced compared with those in pair-fed controls. PPAR expression was down-regulated 3-fold compared with that in pair-fed animals. GPAT expression was also reduced compared with that in both vehicle controls and pair-fed groups.
FIG. 5. Gene expression changes restricted to C75-treated lean mice. Six mice were treated with vehicle () or C75 ( ) every other day. An additional cohort of six mice ( ) was pair-fed to the amounts consumed by the C75-treated animals. After 2-wk treatment, tissues were harvested 4 h following the final dose. Total RNA (0.1 μg) was analyzed by real-time RT-PCR. A, The expression levels of message for ACC, FAS, PPAR, and GPAT were measured in C75-treated lean mice in WAT. B, The expression levels of PPAR and GPAT in liver were measured in C75-treated lean mice. In both tissues, changes unique to C75 treatment were demonstrated. Data are expressed as the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
In the liver of C75-treated mice (Fig. 5B), PPAR expression was also reduced nearly 2-fold compared with that in pair-fed mice. GPAT expression in C75-treated mice was similar to that in controls, but was reduced nearly 2-fold compared with that in the pair-fed group. Taken together, these data show that in lean animals, C75 did not enhance the expression of genes responsible for fatty acid oxidation. Instead, the combined reduction of de novo fatty acid synthesis and GPAT inhibition would probably lead to a net reduction of fatty acids available for triglyceride synthesis.
Alterations in hypothalamic neuropeptide expression in C75-treated DIO and lean mice
Similar to our studies of ob/ob and lean mice acutely treated with C75 (6), there was a reduction of NPY expression after 2 wk of C75 administration compared with that in pair-fed controls (Fig. 6A). In addition, AGRP expression was comparably reduced. Expression of the anorexigenic neuropeptides, POMC and cocaine-amphetamine-related transcript (CART), were increased compared with that in pair-fed animals, with no significant changes in melanin-concentrating hormone (MCH) expression. This hypothalamic profile of reduced orexigenic neuropeptide/increased anorexigenic neuropeptide expression is consistent with the significantly reduced food intake during the 2-wk treatment. In contrast, the hypothalamic profile of the lean mice (Fig. 6B) was notable for no changes in all three orexigenic neuropeptides, with significantly decreased expression of POMC and CART. This is in keeping with the similar food intake in C75-treated and control mice by the end of the 2-wk treatment.
FIG. 6. Effect of C75 on hypothalamic neuropeptide expression in DIO and lean mice. Six mice were treated with vehicle () or C75 ( ) every other day. An additional cohort of six mice ( ) was pair-fed to the amounts consumed by the C75-treated animals. After 2 wk of treatment, tissues were harvested 4 h following the final dose. Total hypothalamic RNA (0.1 μg) was analyzed by real-time RT-PCR. Hypothalamic neuropeptide expression was measured in DIO (A) and lean (B) mice. Data are expressed as the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Discussion
In the present study we quantified hypothalamic neuropeptide expression along with the expression of key genes involved in fatty acid metabolism in peripheral tissues after 2 wk of C75 treatment in DIO and lean mice using real-time RT-PCR. Similar to previously published studies (5, 13), we confirmed that C75 treatment significantly curtailed food intake and increased fatty acid oxidation, leading to dramatic weight loss in DIO mice. In lean mice, significant weight reduction occurred compared with pair-fed controls; however, indirect calorimetry failed to document a significant increase in energy expenditure as fatty acid oxidation during the final 24 h of the study. Although this finding does not rule out increased fatty acid oxidation in the lean animals during C75 administration, clearly, fatty acid oxidation was dramatically increased in DIO mice compared with that in the lean group.
Both C75 treatment and pair-feeding dramatically impacted fatty acid metabolism gene expression in DIO mice. A set of genes showed expression changes common to both the C75-treated and pair-fed groups, consistent with a response to reduced food consumption. This group included dramatic reductions in the lipogenic genes, ACC? and FAS, in both liver and WAT. ACC carboxylates acetyl-CoA to produce malonyl-CoA, the primary substrate for FAS. In liver, the expression of L-CTP-1 and ACO was increased along with that of UCP3 in muscle, consistent with increased fatty acid oxidation. CPT-1 esterifies long-chain acyl-CoAs to carnitine, thus allowing their passage into the mitochondrion for fatty acid oxidation (11, 12); ACO is the rate-limiting enzyme of peroxisomal fatty acid oxidation (26). Although the primary function of UCP3 is unclear, it is up-regulated in muscle as a response to increased fatty acid oxidation (27). Collectively, these findings are consistent with a normal physiological response to food deprivation consisting of decreased lipogenesis and increased fatty acid oxidation.
There were a number of genes whose expression changes were restricted to the C75-treated DIO group, and these most commonly occurred in WAT. Many of these gene expression changes favored oxidation of fatty acid over its incorporation into structural lipids or triglyceride. ACC? was dramatically down-regulated by C75 along with GPAT, whereas L-CPT-1 expression was increased. By reducing ACC? expression, C75 may reduce malonyl-CoA production in the vicinity of L-CPT-1, thus increasing L-CPT-1 activity while concomitantly increasing L-CPT-1 expression. These changes would establish a permissive state for the entrance of fatty acid into the mitochondria for oxidation. GPAT is the initial committed step for fatty acid incorporation into structural lipids or triglycerides (25). C75 inhibition of GPAT expression would further promote fatty acid oxidation by routing fatty acids away from storage and toward transport by the CPT-1 system for oxidation. C75 thus promotes both the shunting of fatty acid to oxidation and the entry of fatty acid into the mitochondria in WAT.
C75 also increased the expression of UCP2 in WAT, liver, and muscle. In addition to promoting uncoupling of mitochondrial oxidative phosphorylation, UCP2 has a role in limiting free radical formation during fatty acid oxidation (28). Through its mitochondrial uncoupling, increased UCP2 expression could allow for increased fatty acid oxidation without the necessity of producing excess ATP.
C75 also affected the expression of both PPAR and PPAR. The PPARs are a group of three nuclear receptor isoforms, PPAR, PPAR, and PPAR, encoded by different genes that regulate a variety of functions related to energy metabolism (29). PPAR is mainly expressed in liver, whereas PPAR is preferentially expressed in WAT. PPAR has been shown to play a critical role in the regulation of cellular uptake, activation, and ?-oxidation of fatty acid (29). Activation of PPAR directly up-regulates the transcription of both types of CPT-1 as well as ACO (30, 31, 32, 33). PPAR serves as a key regulator of adipocyte differentiation and lipid storage (17, 34). In DIO mice, C75 reduced the expression of PPAR in WAT, further promoting the reduction of lipid storage. In the liver, C75 reduced the expression of PPAR despite the increased expression of both L-CPT-1 and ACO. These data suggest that the pattern of gene expression favoring fatty acid oxidation was not due to increased PPAR activity, but was caused by an as yet undetermined mechanism. Accomplishing increased fatty acid oxidation without enhancing PPAR expression could have a particular advantage to cardiac muscle. Increased PPAR expression increases fatty acid transport beyond the capacity of increased fatty acid oxidation, leading to fatty acid deposition in cardiac muscle. This is thought to be the mechanism responsible for diabetic cardiomyopathy (35). Real-time RT-PCR measurements of cardiac muscle after 1 month of C75 treatment failed to show any increase in PPAR expression (data not shown).
In the lean mice, there were no genes with expression changes common to both the C75 and pair-fed groups. A number of genes, however, showed expression changes restricted to C75 treatment. In response to C75, lipogenesis was reduced in WAT, as evidenced by decreased expression of ACC and FAS. Although PPAR expression was reduced, its significance is unknown, because it is not highly expressed in WAT. GPAT expression was reduced in WAT and also in liver along with PPAR. Taken together, these changes indicate a C75-driven reduction in fatty acid synthesis, with a potential for increased fatty acid oxidation due to the reduction of GPAT expression.
Differences between the effects of C75 on DIO and lean mice might be due to different peripheral metabolisms. In DIO mice, C75 increased energy expenditure as fatty acid oxidation manifested as increased VO2 and reduced RER compared with those in pair-fed animals. These data indicate that energy expenditure in DIO mice was mainly from fatty acid oxidation. In lean mice, the diet was high in carbohydrates, consistent with the high RER levels. Although there was no significant increase in energy expenditure with C75, there was a modest reduction in RER compared with pair-fed animals, indicating increased oxidation of fatty acids over glucose. This is in keeping with the gene expression analysis showing that genes involved in fatty acid oxidation were only induced by C75 in DIO mice, not in lean mice. The combination of altered gene expression and the high fat diet in DIO mice is probably responsible for the metabolic differences between lean and DIO mice.
Studies have demonstrated fundamental differences in hypothalamic neuropeptide responses between DIO and lean rodents (36, 37, 38, 39, 40). Consistent with these models, C75 treatment had qualitatively different effects on hypothalamic neuropeptide expression in DIO and lean mice in this study. C75 dramatically inhibited orexigenic neuropeptides (NPY and AGRP) expression and induced anorexigenic neuropeptides (POMC and CART) expression in DIO mice. In lean mice, C75 decreased the expression of anorexigenic neuropeptides (POMC and CART) without changes in the expression of orexigenic neuropeptides (NPY and AGRP). Despite the difference in methodology and number of samples, the overall pattern of expression is similar to that in our prior study (5) and reflects the levels of food consumption. The DIO mice continued to exhibit reduced food intake during the entire duration of treatment, and they displayed a strongly anorexigenic neuropeptide profile. In contrast, by the conclusion of treatment, the lean mice were eating an amount nearly equivalent to controls, and they had a modestly orexigenic hypothalamic profile.
The results of our fatty acid metabolism gene expression analysis advance our understanding of the selectivity of C75 in reducing adipose tissue mass. Conceptually, the dramatic increase in fatty acid oxidation in C75-treated DIO mice would probably require more than competitive stimulation of CPT-1. The gene expression changes in WAT from C75-treated DIO mice increase the expression of enzymes responsible for both the transport of fatty acid into the mitochondria and the shunting of fatty acids away from anabolic pathways to oxidative catabolism. Moreover, the increase in UCP2 expression in liver, WAT, and muscle would allow for increased fatty acid oxidation by mitochondrial uncoupling without requiring superphysiological production of ATP and excessive free radical generation. Interestingly, additional studies have shown that the weight loss effect of C75 treatment persists for nearly 2 wk beyond cessation of therapy (data not shown), also supporting the importance of these gene expression changes for C75 weight maintenance. The exploration of the mechanism of action of C75 serves to further our understanding of the biological consequences of fatty acid synthesis inhibition and fatty acid oxidation stimulation in vivo.
Acknowledgments
We thank members of the FAS Working Group for critical reading of this manuscript.
References
Gale SM, Castracane VD, Mantzoros CS 2004 Energy homeostasis, obesity and eating disorders: recent advances in endocrinology. J Nutr 134:295–298
Must A, Spadano J, Coakley EH, Field AE, Colditz G, Dietz WH 1999 The disease burden associated with overweight and obesity. JAMA 282:1523–1529
Kopelman P 2000 Obesity as a medical problem. Nature 404:635–643
Glazer G 2001 Long-term pharmacotherapy of obesity 2000: a review of efficacy and safety. Arch Intern Med 161:1814–1824
Thupari JN, Kim EK, Moran TH, Ronnett GV, Kuhajda FP 2004 Chronic C75 treatment of diet-induced obese mice increases fat oxidation and reduces food intake to reduce adipose mass. Am J Physiol 287:E97–E104
Loftus T, Jaworsky D, Frehywot G, Townsend CA, Ronnett GV, Lane MD, Kuhajda FP 2000 Reduced food intake and body weight in mice treated with fatty acid synthase inhibitors. Science 288:2379–2381
Kim EK, Miller I, Landree LE, Borisy-Rudin FF, Brown P, Tihan T, Townsend CA, Witters LA, Moran TH, Kuhajda FP, Ronnett GV 2002 Expression of FAS within hypothalamic neurons: a model for decreased food intake after C75 treatment. Am J Physiol 283:E867–E879
Kumar MV, Shimokawa T, Nagy TR, Lane MD 2002 Differential effects of a centrally acting fatty acid synthase inhibitor in lean and obese mice. Proc Natl Acad Sci USA 99:1921–1925
Shimokawa T, Kumar MV, Lane MD 2002 Effect of a fatty acid synthase inhibitor on food intake and expression of hypothalamic neuropeptides. Proc Natl Acad Sci USA 99:66–71
Kim EK, Miller I, Aja S, Landree LE, Pinn M, McFadden J, Kuhajda FP, Moran TH, Ronnett GV 2004 C75, a fatty acid synthase inhibitor, reduces food intake via hypothalamic AMP-activated protein kinase. J Biol Chem 279:19970–19976
McGarry JD, Brown NF 1997 The mitochondrial carnitine palmitoyltransferase system from concept to molecular analysis. Eur J Biochem 244:1–14
Eaton S, Bartlett K, Quant PA 2001 Carnitine palmitoyl transferase I and the control of ?-oxidation in heart mitochondria. Biochem Biophys Res Commun 285:537–539
Thupari JN, Landree LE, Ronnett GV, Kuhajda FP 2002 C75 increases peripheral energy utilization and fatty acid oxidation in diet-induced obesity. Proc Natl Acad Sci USA 99:9498–9502
Woeltje KF, Esser V, Weis BC, Cox WF, Schroeder JG, Liao ST, Foster DW, McGarry JD 1990 Inter-tissue and inter-species characteristics of the mitochondrial carnitine palmitoyltransferase enzyme system. J Biol Chem 265:10714–10719
Weis BC, Esser V, Foster DW, McGarry JD 1994 Rat heart expresses two forms of mitochondrial carnitine palmitoyltransferase I. The minor component is identical to the liver enzyme. J Biol Chem 269:18712–18715
Esser V, Brown NF, Cowan AT, Foster DW, McGarry JD 1996 Expression of a cDNA isolated from rat brown adipose tissue and heart identifies the product as the muscle isoform of carnitine palmitoyltransferase I (M-CPT-I). J Biol Chem 271:6972–6977
Kersten S 2002 Peroxisome proliferator activated receptors and obesity. Eur J Pharmacol 440:223–234
Bocher V, Pineda-Torra I, Fruchart JC, Staels B 2002 PPARs: transcription factors controlling lipid and lipoprotein metabolism. Ann NY Acad Sci 967:7–18
Jezek P 2002 Possible physiological roles of mitochondrial uncoupling proteins: UCPn. Int J Biochem Cell Biol 34:1190–1206
Ledesma A, de Lacoba MG, Rial E 2002 The mitochondrial uncoupling proteins. Genome Biol 3:Reviews3015.1–3015.9
Cha SH, Hu Z, Lane MD 2004 Long-term effects of a fatty acid synthase inhibitor on obese mice: food intake, hypothalamic neuropeptides, and UCP3. Biochem Biophys Res Commun 317:301–308
Ha J, Lee JK, Kim KS, Witters LA, Kim KH 1996 Cloning of human acetyl-CoA carboxylase-? and its unique features. Proc Natl Acad Sci USA 93:11466–11470
Lemberger T, Desvergne B, Wahli W 1996 Peroxisome proliferator-activated receptors: a nuclear receptor signaling pathway in lipid physiology. Annu Rev Cell Dev Biol 12:335–363
Laplante M, Sell H, MacNaul KL, Richard D, Berger JP, Deshaies Y 2003 PPAR- activation mediates adipose depot-specific effects on gene expression and lipoprotein lipase activity: mechanisms for modulation of postprandial lipemia and differential adipose accretion. Diabetes 52:291–299
Dircks LK, Sul HS 1997 Mammalian mitochondrial glycerol-3-phosphate acyltransferase. Biochim Biophys Acta 1348:17–26
Nohammer C, El-Shabrawi Y, Schauer S, Hiden M, Berger J, Forss-Petter S, Winter E, Eferl R, Zechner R, Hoefler G 2000 cDNA cloning and analysis of tissue-specific expression of mouse peroxisomal straight-chain acyl-CoA oxidase. Eur J Biochem 267:1254–1260
Himms-Hagen J, Harper ME 2001 Physiological role of UCP3 may be export of fatty acids from mitochondria when fatty acid oxidation predominates: an hypothesis. Exp Biol Med 226:78–84
Echtay KS, Roussel D, St-Pierre J, Jekabsons MB, Cadenas S, Stuart JA, Harper JA, Roebuck SJ, Morrison A, Pickering S, Clapham JC, Brand MD 2002 Superoxide activates mitochondrial uncoupling proteins. Nature 415:96–99
Berger J, Moller D 2002 The mechanisms of action of PPARs. Annu Rev Med 53:409–435
Brady PS, Marine KA, Brady LJ, Ramsay RR 1989 Co-ordinate induction of hepatic mitochondrial and peroxisomal carnitine acyltransferase synthesis by diet and drugs. Biochem J 260:93–100
Dreyer C, Krey G, Keller H, Givel F, Helftenbein G, Wahli W 1992 Control of the peroxisomal ?-oxidation pathway by a novel family of nuclear hormone receptors. Cell 68:879–887
Tugwood JD, Issemann I, Anderson RG, Bundell KR, McPheat WL, Green S 1992 The mouse peroxisome proliferator activated receptor recognizes a response element in the 5' flanking sequence of the rat acyl CoA oxidase gene. EMBO J 11:433–439
Mascaro C, Acosta E, Ortiz JA, Marrero PF, Hegardt FG, Haro D 1998 Control of human muscle-type carnitine palmitoyltransferase I gene transcription by peroxisome proliferator-activated receptor. J Biol Chem 273:8560–8563
Gerhold DL, Liu F, Jiang G, Li Z, Xu J, Lu M, Sachs JR, Bagchi A, Fridman A, Holder DJ, Doebber TW, Berger J, Elbrecht A, Moller DE, Zhang BB 2002 Gene expression profile of adipocyte differentiation and its regulation by peroxisome proliferator-activated receptor- agonists. Endocrinology 143:2106–2118
Finck BN, Lehman JJ, Leone TC, Welch MJ, Bennett MJ, Kovacs A, Han X, Gross RW, Kozak R, Lopaschuk GD, Kelly DP 2002 The cardiac phenotype induced by PPAR overexpression mimics that caused by diabetes mellitus. J Clin Invest 109:121–130
Bergen HT, Mizuno T, Taylor J, Mobbs CV 1999 Resistance to diet-induced obesity is associated with increased proopiomelanocortin mRNA and decreased neuropeptide Y mRNA in the hypothalamus. Brain Res 851:198–203
Levin BE, Dunn-Meynell AA 2000 Sibutramine alters the central mechanisms regulating the defended body weight in diet-induced obese rats. Am J Physiol 279:R2222–R2228
Ziotopoulou M, Mantzoros CS, Hileman SM, Flier JS 2000 Differential expression of hypothalamic neuropeptides in the early phase of diet-induced obesity in mice. Am J Physiol 279:E838–E845
Wang H, Storlien LH, Huang X 2002 Effects of dietary fat types on body fatness, leptin, and ARC leptin receptor, NPY, and AgRP mRNA expression. Am J Physiol 282:E1352–E1359
Rohner-Jeanrenaud F, Craft LS, Bridwell J, Suter TM, Tinsley FC, Smiley DL, Burkhart DR, Statnick MA, Heiman ML, Ravussin E, Caro JF 2002 Chronic central infusion of cocaine- and amphetamine-regulated transcript (CART 55–102): effects on body weight homeostasis in lean and high-fat-fed obese rats. Int J Obes Relat Metab Disord 26:143–149(Yajun Tu, Jagan N. Thupar)