Short-term resistance to diet-induced obesity in A/J mice is not associated with regulation of hypothalamic neuropeptides

John W. Bullen, Jr,1 Mary Ziotopoulou,1 Linda Ungsunan,1 Jatin Misra,2 Ilias Alevizos,2 Efi Kokkotou,3 Eleftheria Maratos-Flier,3 Gregory Stephanopoulos,2 and Christos S. Mantzoros1

1Division of Endocrinology, Beth Israel Medical Center, Harvard Medical School, and 3Joslin Diabetes Center and Harvard Medical School, Boston 02215; and 2Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Submitted 9 March 2004 ; accepted in final form 4 May 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To investigate the mechanisms underlying long-term resistance of the A/J mouse strain to diet-induced obesity, we studied, over a period of 4 wk, the expression of uncoupling proteins in brown adipose tissue and the expression of hypothalamic neuropeptides known to regulate energy homeostasis and then used microarray analysis to identify other potentially important hypothalamic peptides. Despite increased caloric intake after 2 days of high-fat feeding, body weights of A/J mice remained stable. On and after 1 wk of high-fat feeding, A/J mice adjusted their food intake to consume the same amount of calories as mice fed a low-fat diet; thus their body weight and insulin, corticosterone, free fatty acid, and glucose levels remained unchanged for 4 wk. We found no changes in hypothalamic expression of several orexigenic and/or anorexigenic neuropeptides known to play an important role in energy homeostasis for the duration of the study. Uncoupling protein-2 mRNA expression in brown adipose tissue, however, was significantly upregulated after 2 days of high-fat feeding and tended to remain elevated for the duration of the 4-wk study. Gene array analysis revealed that several genes are up- or downregulated in response to 2 days and 1 wk of high-fat feeding. Real-time PCR analysis confirmed that expression of the hypothalamic IL-1 pathway (IL-1{beta}, IL-1 type 1 and 2 receptors, and PPM1b/PP2C-{beta}, a molecule that has been implicated in the inhibition of transforming growth factor-{beta}-activated kinase-1-mediated IL-1 action) is altered after 2 days, but not 1 wk, of high-fat feeding. The role of additional molecules discovered by microarray analysis needs to be further explored in the future.

neuropeptides; energy homeostasis; uncoupling protein-2; interleukin-1; microarray


THE EPIDEMIC OF OBESITY in Western populations is associated with overconsumption of high-fat diets. The exact mechanism by which certain people (34) and certain strains of rodents (44) are susceptible to high-fat diet-induced obesity (DIO), whereas others are relatively resistant to its effects, is the subject of intensive investigation.

The most thoroughly studied experimental model of DIO is the C57Bl/6J mouse strain, whereas one of the most commonly studied DIO-resistant models is the A/J mouse strain (33, 3840). Investigation of the physiological responses of neuroendocrine factors, hypothalamic neuropeptides, and uncoupling proteins (UCPs) to high-fat feeding in these strains of mice could extend our understanding of the pathogenesis of DIO in humans, leading to potentially important therapeutic advances. Although several studies have used the DIO-sensitive C57Bl/6J strain to examine the effect of diet composition on hypothalamic neuropeptides and/or neuroendocrine factors important in energy homeostasis, only one study has focused on the hypothalamic expression of neuropeptides in the DIO-resistant A/J strain (1). This long-term study (1) reported that resistance to DIO in A/J mice after 14 wk on a high-fat diet is associated with increased proopiomelanocortin (POMC) and decreased neuropeptide Y (NPY) hypothalamic mRNA levels. No previous study has examined alterations of hypothalamic neuropeptide levels in response to high-fat feeding of A/J mice over a shorter period of time, during which the physiological adaptations of food intake occur, and/or longitudinally at several points over time. To gain further insight into the mechanisms underlying the physiological response of the A/J strain exposed to a high-fat diet, we studied longitudinally, over a 4-wk period, the hypothalamic expression of neuropeptides known to be implicated in energy homeostasis and/or leptin action [NPY, Agouti-related protein (AgRP), melanin-concentrating hormone (MCH), POMC, and orexin] as well as the expression of UCPs responsible for energy dissipation in brown adipose tissue (BAT). In addition, we investigated the expression of three potential molecules previously shown to regulate leptin action in the hypothalamus [suppressor of cytokine signaling (SOCS)-3, protein inhibitor of activated STAT3 (PIAS-3), and protein tyrosine phosphatase (PTP)-1b]. SOCS-3 and PIAS-3 have been implicated in the induction of leptin resistance, and PTP-1b has been implicated in regulating insulin and leptin signaling by targeting Jak2 in the hypothalamus (6, 47).

Of all the initial molecules investigated, only UCP2 exhibited significant upregulation in BAT of A/J mice in response to 2 days, 3 wk, and 4 wk of high-fat feeding. Although this could contribute to the development of resistance to DIO in A/J mice, we hypothesize that other factors are also likely to contribute to this response. We thus performed microarray analysis on hypothalamic RNA from A/J mice at the 2-day and 1-wk time points to assess whether other molecules that were not previously shown to be associated with the direct regulation of energy homeostasis are involved in the apparent regulation of food intake in A/J mice fed a high-fat diet.

We hereby show that exposure to a high-fat diet for 2 days is associated with decreased expression of molecules in the IL-1{beta} pathway [IL-1{beta} and IL-1{beta} receptors (IL-1R1 and IL-1R2)] and may be contributing to increased food intake during this period. In contrast, the upregulation of UCP2 in BAT may be of importance in determining why there is resistance to DIO in A/J mice for 2 days to 4 wk. Finally, the role of additional molecules revealed by microarray analysis needs to be confirmed and further explored in the future.


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

A/J mice were obtained from Jackson Laboratories (Bar Harbor, ME) at 3–5 wk of age. All animals were housed in a temperature-controlled room maintained at 25°C with a 12:12-h light-dark cycle (lights on from 0630 to 1830) for an acclimatization period of ≥1 wk. All animals had free access to water and Purina Rodent Chow 5008 [16.7% of calories from fat, 56.4% of calories from carbohydrate, and 26.8% of calories from protein (wt/wt), 3.5 kcal/g]. Two separate cohorts of animals were used to produce the data presented herein: the first for experiments described under study 1 and the second for experiments described under study 2.

Study 1 investigated the role of hypothalamic factors that are generally considered to play an important role in regulating energy homeostasis and/or leptin action [NPY, AgRP, MCH, MCH receptor (MCH-R), POMC, orexin, SOCS-3, and PIAS]. In addition, microarray analysis was used to discover other potentially important molecules that are responsible for resistance to DIO in A/J mice. Study 2 served to independently validate the expression of molecules discussed above and to explore the role of additional molecules that are known to be implemented in the regulation of energy homeostasis (PTP-1b, UCP1, and UCP2) or were revealed by the microarray analysis of animals in study 1 [corticotropin-releasing hormone (CRH) receptors (CRH-R1 and CRH-R2), IL-1{beta}, IL-1R1, IL-1R2, and magnesium-dependent phosphatase (PPM1b)].

Both cohorts were housed under identical conditions with the exception of the use of different high- and low-fat diets. Validation studies were performed to disprove the possibility of diet-specific data (see RESULTS). For study 1, all animals were individually housed for 1 mo before being weight matched and divided into eight groups (n = 8/group). Four groups were fed a high-fat diet and four groups were fed a low-fat diet for 2 days, 1 wk, 2 wk, or 4 wk. High- and low-fat diets were obtained from Research Diets [no. D12451 [GenBank] : 44.9% of calories from fat, 35.1% of calories from carbohydrate, 20% of calories from protein (wt/wt), 4.73 kcal/g; and no. D12450B: 10% of calories from fat, 70.0% of calories from carbohydrate, 20.0% of calories from protein (wt/wt), 3.85 kcal/g, respectively] as previously described (48).

For study 2, all animals were individually housed for 1 wk before being weight matched and divided into eight groups (n = 5/group). Similar to study 1, four groups of animals were fed a high-fat diet and four groups were fed a low-fat diet for 2 days, 1 wk, 3 wk, and 4 wk. The low-fat diet (Purina Rodent Chow 5008) contained 16.7% of calories from fat. The high-fat diet (Harlan Teklad TD 88137) contained 42.16% of calories from fat, 42.81% of calories from carbohydrate, and 15.02% of calories from protein (4.53 kcal/g).

All animals were handled in accordance with the principles and guidelines established by the National Institutes of Health and the Institutional Animal Care and Use Committee of Beth Israel Deaconess Medical Center.

Experimental Procedures

An analytic balance was used to measure body and food weights at 9:30 AM daily for 2 days, 1 wk, 2 wk, 3 wk, or 4 wk. Because inspection of the cages revealed no detectable spillage of food, differences in food weights were assumed to represent the amount of food intake per day. Animals were studied in the fed state to avoid altered neuropeptide levels due to fasting. Mice were killed by CO2 narcosis between 8:00 and 9:30 AM. Hypothalami and BAT were collected, immediately snap frozen in liquid nitrogen, and stored at –80°C until analyzed for neuropeptide gene expression. Blood was collected by cardiac puncture and immediately assayed for blood glucose levels (One Touch Profile Blood Glucose Meter, Lifescan, Milpitas, CA). Serum was separated from blood and stored at –80°C for further analysis of leptin, insulin, corticosterone, and nonesterified fatty acids (FFA) as previously described (48). Insulin, corticosterone, and leptin were assayed in duplicate by RIA (Mouse Leptin and Rat Insulin Kit, Linco Research Institute, St. Louis, MO; and Rat Corticosterone Kit, ICN, Costa Mesa, CA). FFA were assayed in duplicate by an enzymatic colorimetric method (Waco Chemicals, Richmond, VA).

Preparation and Quantification of NPY, AgRP, POMC, Orexin, MCH, MCH-R, SOCS-3, PIAS-3, CRH-R1, CRH-R2, PTP-1b, UCP1, UCP2, PPM1b, IL-1{beta}, IL-1R1, and IL-1R2 Gene Expression

Hypothalamic and BAT total RNA was purified using the STAT-60 method (TEL-TEST, Friendswood, TX). cDNA synthesis was performed from 1 µg of total RNA by use of the BD Biosciences (Clontech) Advantage RT for PCR kit and methods (BD Biosciences, Palo Alto, CA). PCR amplifications of NPY, AgRP, POMC, orexin, SOCS-3, and PIAS-3 were carried out using mouse specific primers as previously described (3, 8, 48).

CRH-R1 and CRH-R2 were amplified using mouse specific CRH-R1 and CRH-R2 primers (45). Mixtures were subjected to 30 cycles of amplification (denaturation at 96°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 2 min). PTP-1b was amplified using the following mouse specific PTP-1b primers: 5'-CCCGGCCACCCAAACGCACACT-3' (sense) and 5'-GACGCCGCAGACCGCATCCTAAGC-3' (antisense). PTP-1b mixtures were subjected to 34 cycles of amplification (denaturation at 96°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 2 min). UCP1 and UCP2 were amplified using the following mouse specific primers: 5'-TGTTGGAATGACAGGAGCTG-3' (sense) and 5'-TCCTGGTGTATGGGCTATGG-3' (antisense) for UCP1 and 5'-TAGTGCGCACCGCAGCC-3' (sense) and 5'-AGCTCATCTGGCGCTGCAG-3' (antisense) for UCP2. For UCP1 and UCP2, mixtures were subjected to 22 cycles of amplification (denaturation at 96°C for 1 min, annealing at 50°C for 1 min, and extension at 72°C for 1.5 min). Semiquantitative RT-PCR analysis of IL-1{beta} was attained using mouse specific IL-1{beta} primers (3). IL-1{beta} mixtures were subjected to 30 cycles of amplification (denaturation at 96°C for 1 min, annealing at 50°C for 1 min, and extension at 72°C for 1 min). Briefly, each 50-µl PCR was carried out with 5.0 µl of cDNA as the template. Assay conditions consisted of 10 mmol/l Tris·HCl (pH 8.8), 50 mmol/l KCl, 1.5 mmol/l MgCl2, 0.01% gelatin, 0.2 mmol/l dNTPs, 20 pmol of each primer, 2.5 U of Taq polymerase (Stratagene, La Jolla, CA), and 0.5 µl of [32P]dCTP (29.6 TBq/mmol, 18.5 MBq/ml; DuPont-NEN, Boston, MA). Each mixture was overlaid with 50 µl of mineral oil and subjected to an initial denaturation period of 96°C for 2–4 min. Twenty microliters of PCR product were loaded per lane on 5% Tris-borate-EDTA polyacrylamide gels. Electrophoresis was carried out as previously described (3, 48). Gels were then transferred to and dried on 3M filter paper and subjected to 32P quantitation by PhoshorImager analysis (Molecular Dynamics, Sunnyvale, CA).

Reported gene expression is represented by the mean values (from arbitrary units calculated by the radioactive intensity as determined by phosphor imaging) of each group normalized to their respective {beta}-actin housekeeping gene values. Each measurement is expressed as a percent change in relation to the values of the low-fat diet group. Quantitative RT-PCR analysis of IL-1{beta}, IL-1R1, and IL-1R2 was attained using mouse specific primers provided by Assays on Demand (PE Biosystems, Foster City, CA). PPM1b was amplified using mouse specific primers designed by Assays by Design (PE Biosystems). PPM1b, MCH and MCH-R (SLC-1) were amplified along with GAPDH (Taqman rodent GAPDH control reagents, PE Biosystems) as previously described (23). RNA samples were analyzed in triplicate using the ABI Prism 7700 sequence detection system (PE Biosystems). Results of PPM1b, MCH, and MCH-R expression were normalized by GAPDH values and then expressed as percent change in relation to the low-fat diet values. GAPDH expression remained unaltered under the different treatments of the animals. IL-1{beta}, IL-1R1, and IL-1R2 were amplified along with 18S (Taqman rodent ribosomal RNA control reagents, PE Biosystems). cDNA samples were subjected to an initial denaturation period of 95°C for 10 min followed by 40 cycles at 95°C for 30 s and 60°C for 1 min. cDNA samples were analyzed in triplicate using the Stratagene Mx4000 Multiplex Quantitative PCR System. Expression of 18S remained unaltered under the different treatment regimens.

Microarrays

Oligonucleotide library and printing. The Mouse Genome Oligo Set (Version 1, Operon Qiagen, Alameda, CA) was used for the creation of DNA microarrays. The set contains 13,500 Mus musculus genes. The set was resuspended in 30 µl of RNase- and DNase-free 3x SSC for a final concentration of 20 µM. The set was printed on GAPS II-coated bar-coded slides (Corning, Corning, NY) with use of the Versarray Chipwriter Pro microarraying robot (Bio-Rad Laboratories, Hercules, CA). Each oligonucleotide was spotted in triplicate. Printing quality was assessed by SYBR II staining (Molecular Probes, Eugene, OR).

Variability of microarray validation. The microarrays have been extensively validated for intra- and interarray variability. A mean coefficient of variation of 20% was observed on the basis of repeated experiments. This implies that ratios >1.4 and <0.6 can be considered significant at the 95% confidence limit. In addition, experiments were conducted to ensure that differential expression could be detected. On a test array, total RNA from healthy skeletal muscle tissue and testes tissue were hybridized against each other. On a control array, skeletal muscle RNA was hybridized against itself. On the basis of the cutoffs established above, ~35% of the genes were differentially expressed in the test array, as opposed to only 6% in the control array, as expected given the 95% confidence limit used here. Several of the discriminatory genes found in the test arrays, such as actins and troponins, were related to skeletal muscle.

Control RNA. Control RNA for all the hybridizations was derived by pooling RNA from 20 mice from the following tissues: hypothalamus, liver, skeletal muscle, brown fat, white fat, kidney, adrenal gland, testes, ovary, heart, and lung.

Labeling and hybridization. Ten micrograms of RNA were used for the analysis of the control and samples. Samples (in duplicate) from each time point and diet were profiled. Hence, because hypothalamic samples of animals fed a high- and a low-fat diet for 2 days and 1 wk were used, a total of 10 samples were profiled. The labeled cDNA synthesis took place as follows: 2 µl of oligo(dT)18–20 primer (Invitrogen, Carlsbad, CA) were added to each sample, and mixtures were heated at 70°C for 10 min and then incubated for 2 min on ice. Subsequently, 2.0 µl of 10x Cy3 dCTP control (Perkin-Elmer, Boston, MA) and 10x Cy5 dCTP (Perkin-Elmer) were added; then 2 µl of 10x dNTPs (Invitrogen), 2 µl of 100 mM DTT (Invitrogen), 4 µl of 5x first-strand buffer (Invitrogen), and 2 µl of Superscript II (Invitrogen) were added. The final mixture was incubated at 42°C for 2 h. On completion of reverse transcription, 1.5 µl of 1 N NaOH were added to each sample, and the samples were incubated at 65°C for 10 min. After incubation, 1.5 µl of 1 N HCl were added to each sample to neutralize NaOH. The Cy3 and Cy5 samples were combined and purified from the unincorporated dyes, nucleotides, and enzymes with the Qiagen QIAquick nucleotide removal kit. The samples were then concentrated and resuspended in 20 µl of warm GlassHyb hybridization buffer (Clontech, Franklin Lakes, NJ) and applied on the microarray slides. A coverslip was carefully placed on top of the slides to ensure the absence of bubbles under the coverslip. The slides were subsequently sealed in Corning hybridization chambers and incubated at 55°C for a total of 12 h. At the end of hybridization, the coverslip was removed and washed in a 1x SSC-1% SDS solution for 5 min at room temperature and washed for 5 min in 0.2x SSC and for 5 min in 0.1x SSC. The slides were then placed in a sterile Falcon tube and dried by spinning at 500 g for 3 min.

Scanning. The dried slides were scanned in the GenePix 4000B microarray scanner (Axon, Union City, CA) and analyzed with the GenePix Pro (Axon) acquisition and analysis software.

Discriminatory gene identification. t-Test analysis was applied to our total gene set to obtain a preliminary list of discriminatory genes. These genes were then ranked on the basis of the number of data points available for each gene, and the significance of the gene was based on the t-test. The significance of a gene is the probability of choosing that particular gene as discriminatory on the basis of random chance. Thus the smaller the level of significance and the greater the number of data points observed for the gene, the higher the reliability of the gene as a discriminator and the higher is the rank given to the gene.

Analysis. On the basis of the observed coefficient of variation of 20%, a 95% confidence limit would imply that genes that were beyond the 1.4- and 0.6-fold levels are significantly up- or downregulated, respectively. These genes were ranked by the magnitude of the mean fold changes for 2 days and 1 wk after high-fat feeding.

Calculations and Statistical Analysis

Values are means ± SE. Statistical significance was assessed by ANOVA with post hoc tests by use of the Statview program (Abacus, CA). Differences were considered significant at two-tailed P < 0.05.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effects of High- and Low-Fat Feeding on Body Weight and Caloric Intake in A/J Mice: Studies 1 and 2

2-Day experiment. Changes in body weight and caloric intake were assessed in response to 2 days of high- and low-fat feeding. After 2 days, final weights and food intake did not differ between the two groups (Table 1). In studies 1 and 2, however, the high-fat diet-fed group had increased cumulative caloric intake by ~30% compared with the low-fat diet-fed group over the 2-day period (Table 1).


View this table:
[in this window]
[in a new window]
 
Table 1. Effects of high- and low-fat feeding of A/J mice on body weight and cumulative food and caloric intake: studies 1 and 2

 
1-Week experiment. After 1 wk, final weights remained unchanged, but cumulative caloric intake was no longer different between the two groups (Table 1). Given that cumulative caloric intake in the high-fat diet-fed group was increased after 2 days, a compensatory decrease in the food consumption of this group must have occurred in the interim, resulting in similar cumulative caloric intakes for the high- and low-fat diet-fed groups after 1 wk.

2-, 3-, and 4-Week experiment. Data at 2, 3, and 4 wk showed results similar to those from the 1-wk experiment. No significant differences in body weight or cumulative caloric intake were found between the two groups (Table 1). Thus mice fed the high-fat diet begin and continue to adjust their food intake after 2 days on a high-fat diet so as to consume the same calories as the low-fat group over the 4-wk time period.

Effects of High-Fat Feeding on Circulating Hormone and FFA Concentrations in A/J Mice: Studies 1 and 2

Leptin. Serum leptin levels in A/J mice were similar between the two groups after 2 days on the diets: 9.07 ± 0.74 and 10.44 ± 1.08 ng/ml in low- and high-fat diet-fed mice, respectively. However, after 1 wk, leptin levels in the high-fat group were significantly higher (11.14 ± 0.78 ng/ml, P < 0.05 vs. low fat fed mice) than corresponding levels in the low-fat group (8.80 ± 0.74 ng/ml). Similar results were obtained at the 1-wk time point of study 2 (data not shown). Although the same magnitude of difference in leptin levels between the high- and low-fat groups persisted at the 2-wk (11.15 ± 0.95 and 8.85 ± 0.70 ng/ml, respectively, P = 0.07) and 4-wk time points (11.42 ± 1.27 and 8.53 ± 1.41, respectively, P = 0.15) because of increased variability, these differences failed to achieve statistical significance.

Corticosterone, insulin, glucose, and FFA. Serum corticosterone, insulin, glucose, and FFA levels were not significantly different between the high- and low-fat groups for all time points (data not shown).

Effects of High-Fat Feeding on Expression of Hypothalamic Neuropeptides and UCPs

Orexigenic peptides: NPY, AgRP, orexin, and MCH. Measurements at 2 days and 1, 2, and 4 wk did not show any differences in NPY expression between the high- and low-fat diet-fed groups (data not shown). Similarly, no differences were observed in AgRP, orexin, MCH, or MCH-R expression between high-and low-fat groups at all time points (data not shown).

Anorexigenic factors: POMC, IL-1{beta}, IL-1R1, and IL-1R2. POMC mRNA expression in high-fat diet-fed mice was not significantly different from that in low-fat diet-fed mice at all respective time points (data not shown). There was an ~58% decrease in IL-1{beta} expression (100.0 ± 21.0% vs. 42.1 ± 7.6% in low- vs. high-fat diet-fed mice, P < 0.05), an ~38% decrease in IL-1R1 expression (100.0 ± 4.3% vs. 62.6 ± 8.4% in low- vs. high-fat diet-fed mice, P < 0.05), and an ~23% decrease in IL-1R2 expression (100.0 ± 7.3% vs. 77.0 ± 4.8% in low- vs. high-fat diet-fed mice, P < 0.05) after 2 days of high-fat feeding, but all expression levels returned to baseline levels after 1 wk (data not shown).

Regulators of leptin and/or insulin action: SOCS-3, PTP-1b, and PIAS-3. Hypothalamic SOCS-3 mRNA expression in high-fat diet-fed mice was not significantly different from that in low-fat diet-fed mice at the 2-day time point (data not shown). A significant increase in SOCS-3 mRNA expression was observed in the high-fat diet-fed group at the 1-wk time point, when serum leptin levels were also increased: 100.0 ± 9.2% vs. 133.7 ± 11.6% in low- vs. high-fat diet-fed mice (P < 0.05). Consistent with the tendency toward normalization of leptin levels at later time points, SOCS-3 expression levels returned to baseline after 2 wk and continued to remain similar in expression to the low-fat diet-fed group after 4 wk (data not shown). PIAS-3 and PTP-1b mRNA expression was unchanged for the entire experimental period (data not shown).

UCP1, UCP2, and PPM1b. There were no significant differences in UCP1 mRNA expression in BAT of A/J mice at all time points (data not shown). UCP2 BAT mRNA expression was significantly upregulated after 2 days of high-fat feeding: 100.0 ± 18.6% vs. 174.6 ± 30.1% in low- vs. high-fat diet-fed mice (P < 0.05). Although there were no apparent differences in UCP2 expression after 1 wk of high-fat feeding, UCP2 expression returned to levels comparable to the 2-day time point at the 3- and 4-wk time points: 100.0 ± 26.1% vs. 148.3 ± 15.8% (P = 0.10) at 3 wk and 100.0 ± 13.4% vs. 158.2 ± 27.5% (P = 0.09) at 4 wk. Despite an ~50% increase in expression at both of these time points, these data failed to reach statistical significance, mostly as a result of increased variability and a small sample size (n = 5). Hypothalamic PPM1b mRNA expression was significantly upregulated after 2 days (100.0 ± 5.8% vs. 127.0 ± 6.1% in low- vs. high-fat diet-fed mice, P < 0.01) but not 1 wk of high-fat feeding (data not shown).

"High-energy diet" vs. "diet-specific" response. As explained in MATERIALS AND METHODS, in addition to the analysis of appetite-regulating factors measured in study 1 (NPY, AgRP, POMC, MCH, orexin, MCH-R, SOCS-3, and PIAS-3), study 2 was performed for the analysis of additional peptides (CRH-R1, CRH-R2, PTP-1b, UCP1, and UCP2). Because two different sets of high- and low-fat diets were used for study 1 and study 2, we also performed validation experiments at the 1-wk time point to disprove the possibility of diet-specific effects in gene expression. In agreement with study 1, there were no differences in NPY, AgRP, POMC, MCH, orexin, or MCH-R hypothalamic mRNA expression at 1 wk in animals from study 2 (data not shown). In addition, similar to study 1, hypothalamic SOCS-3 mRNA expression was significantly upregulated at 1 wk in response to high-fat feeding (100.0 ± 0.076% vs. 119.36 ± 7.52% in low- vs. high-fat diet-fed mice, P = 0.043), indicating that the observations are robust, regardless of the high-fat diet used, and thus are independent of the specific diet type used.

Microarray Analysis of Hypothalamic mRNA Expression

All the genes analyzed by RT-PCR discussed above were not present in Version 1 of the Operon Mouse Genome Oligo Set or, if present (e.g., CRH-R, upregulated: +0.51 after 2 days and +0.76 after 1 wk), failed to rank high enough to be considered significantly discriminatory at P < 0.05. Although CRH-R was not above the +1.4 cutoff to be considered significantly upregulated because of the CRH system's considerable anorexic influence on food intake (17), we also used RT-PCR to measure these receptors' hypothalamic mRNA expression to further confirm the microarray data. CRH-R1 and CRH-R2 mRNA expression was unchanged at all time points by RT-PCR and, therefore, is in agreement with the microarray data presented above.

Differentially expressed genes after 2 days and 1 wk. The genes displayed in Table 2 are ranked by the magnitude of their mean ratio fold differences compared with controls for both time points (2 days and 1 wk) and represent the top 50 most "reliable" genes (all unknown RIKEN cDNA clones are excluded). There was an approximately twofold change in PPM1b mRNA expression in response to 2 days and 1 wk of high-fat feeding (Table 2), but RT-PCR validation revealed only a significant upregulation at 2 days, as described above. Similarly, IL-1R1 mRNA expression was decreased by ~1.5-fold in response to high-fat feeding for the same two time points and is further validated by RT-PCR as described above.


View this table:
[in this window]
[in a new window]
 
Table 2. Hypothalamic gene expression in response to 2 days and 1 wk of high-fat feeding in DIO-resistant A/J mice

 
Differentially expressed genes after 2 days, but not 1 wk, or after 1 wk, but not 2 days. Asb5 and Ugt1a6 were downregulated after 2 days of high-fat feeding but returned to normal expression levels at 1 wk (Table 2). Fabp7, NeuroD3, RassF5, and Mrps5 exhibited a decrease in mRNA expression after 1 wk of high-fat feeding but were unchanged after 2 days (Table 2). The differential expression of these molecules remains to be validated.


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We previously demonstrated that hypothalamic expression of two known orexigenic peptides, NPY and AgRP, was decreased by ~30% and ~50%, respectively, by high-fat feeding of DIO-prone C57Bl/6J mice for 2 days and returned to normal after 1 wk of high-fat feeding (48). Hypothalamic expression of POMC, a known anorexigenic peptide, was increased by ~76% after 2 wk of high-fat feeding in C57Bl/6J mice (48), but not after 14 wk (1). In contrast, it has been demonstrated that high-fat feeding of A/J mice for 14 wk is associated with increased POMC and decreased NPY hypothalamic mRNA levels, but how A/J mice regulate food intake acutely in response to high-calorie diets remains unknown. We hypothesized that this occurs through even more pronounced alterations of key hypothalamic molecules involved in food intake regulation and, thus, measured the expression of several neuropeptides important in energy homeostasis.

First, we investigated the melanocortin pathway, an anorexigenic pathway (10, 21, 27), and NPY, one of the most important orexigenic neuropeptides implicated in food intake regulation (21). We have shown here that AgRP, NPY, and POMC mRNA levels in A/J mice are not altered during the first 4 wk of high-fat feeding, suggesting that other mechanisms must be responsible for the observed compensatory decreases in caloric intake. To gain further insights into how A/J mice resist DIO, we then investigated additional neuropeptides important in regulating food intake and energy balance, such as MCH and MCH-R (32), orexin (35), CRH-R1 and CRH-R2 (19), and PTP-1b (22). MCH expression is increased by fasting (27), and its intracerebroventricular administration increases food intake (24), whereas mice with targeted ablation of MCH are hypophagic and lean (36). We observed no changes in hypothalamic MCH or MCH-R levels, nor did we find any variation in hypothalamic orexin mRNA expression in response to low- or high-fat feeding of A/J mice at any of the time points studied. Although orexins A and B were initially proposed to promote feeding after intracerebroventricular administration (35), later studies showed that orexins were more likely to affect sleep or other behavioral activities (5) than food intake (41).

CRH, a potent anorexigenic peptide, acting downstream of leptin (19), acts by activating CRH-R1 and CRH-R2 (16). We observed no short-term differences in CRH-R1 or CRH-R2 hypothalamic mRNA expression in this experiment. We then examined PTP-1b, a negative regulator of insulin, which has also been implicated in inhibiting leptin signaling (22, 42, 48). Although PTP-1b-knockout mice are protected against insulin resistance and obesity (7), we detected no differences in PTP-1b expression at any of the time points studied herein, excluding changes of this molecule as a potential mechanism responsible for DIO resistance in A/J mice. Therefore, we detected no short-term changes in the hypothalamic neuropeptides mentioned above, which could have possibly supported the differential response of A/J mice to high-fat diets.

Similar to previous studies, leptin levels of A/J mice in this study were also increased by 25% after 1 wk of high-fat feeding and tended to remain elevated, albeit nonsignificantly, for 4 wk. In addition, previous studies have shown that A/J mice fed a high-fat diet for 4 and 8 wk are fully responsive to intracerebroventricular injections of leptin, whereas C57Bl/6J mice are not (30); therefore, compensatory increases in serum leptin levels in response to high-fat feeding could be of significance. To assess the potential physiological significance of increased serum leptin levels in A/J mice fed a high-fat diet for 4 wk, we examined whether expression of hypothalamic neuropeptides and inhibitors of leptin signaling were altered during the first 4 wk of high-fat feeding. The expression of negative regulators of the JAK-STAT pathway, such as SOCS-3 and PIAS-3 (2, 18, 37), is upregulated by leptin, resulting in inhibition of leptin signaling and activation of STAT, and provides an in vivo indicator of direct leptin stimulation of neurons expressing the long form of the leptin receptor (2). Although PIAS-3 mRNA levels remained unchanged during the period studied, SOCS-3 expression was significantly upregulated after 1 wk on the high-fat diet, when leptin levels were also increased. At 2 and 4 wk, when leptin expression tended to return toward baseline levels, SOCS-3 expression also returned to baseline levels. In this respect, our results suggest that, despite A/J mice exhibiting slightly elevated leptin levels, they retain leptin sensitivity and, therefore, may resist DIO by increasing nonshivering thermogenic activity in BAT.

Nonshivering thermogenesis in BAT serves to regulate body temperature and body weight through UCPs, which, by uncoupling the process of mitochondrial respiration from oxidative phosphorylation, diminish the production of ATP and yield dissipative heat (11). High-fat feeding to rats for 1 wk induces UCP1 gene expression in BAT, which may decrease fat synthesis and increase thermogenesis (15). UCP1 mRNA levels in BAT are increased after 10 and 16 wk of high-fat feeding in A/J mice, but not in C57Bl/6J mice. Serum leptin levels and UCP2 expression in white adipose tissue (WAT) of A/J mice are higher than baseline measurements and measurements in C57B6/J mice after 2, 10, and 16 wk of high-fat feeding (43), suggesting that retention of leptin responsiveness in A/J mice, as indicated by increased UCP mRNA expression in response to leptin administration (30), is an important component of the ability of A/J mice to mount a robust adaptive thermogenic response and resist obesity. These findings may be strain specific, however, for they are not consistent in all DIO-prone or DIO-resistant mouse models. High-fat feeding of DIO-prone AKR/J mice for 4 wk results in decreased UCP1 expression and increased UCP2 expression in BAT and WAT, an effect that is, for the most part, absent in DIO-resistant SWR/J mice (31). In agreement with previous data in A/J mice (43), UCP2 mRNA levels in BAT were significantly increased after 2 days and remained elevated at 3 and 4 wk. Thus it could be proposed that the ability of A/J mice to resist DIO, at least in the short term, may partially be associated with a strain-specific increase in UCP2 expression in WAT and BAT, whereas the downregulation of UCP1 may predispose AKR/J mice to develop obesity. An investigation of not only UCP expression but also UCP protein regulation in BAT, as well as direct measurements of energy expenditure, at earlier and later time points in various DIO-prone/resistant mouse models, is needed to fully elucidate the role of UCPs in long-term resistance to DIO in A/J mice.

Past studies have shown that C57Bl/6J mice do not exhibit any significant differences in insulin, corticosterone, or FFA levels in response to 2 days or 1 wk of high-fat feeding (48). Similarly, we found no significant differences in insulin, glucose, corticosterone, or FFA levels in A/J mice during any of the time points studied here. Because, in contrast to noticeable short-term regulation of these neuropeptides in DIO-prone C57Bl/6J mice, there is a lack of short-term regulation of candidate orexigneic and/or anorexigenic neuropeptides, as well as metabolic hormones in A/J mice, one could hypothesize that there must be some other mechanism regulating food, and resultant caloric, intake in A/J mice. Although there is evidence of increased energy expenditure by increased UCP2 expression in BAT, as stated above, changes in UCP expression cannot fully account for decreases in food intake after 1 wk on high-fat diet to levels similar to those observed on a low-fat diet that is followed by approximately equal caloric intake over time.

To gain further insight as to what other hypothalamic orexigenic or anorexigenic factors may be involved in the apparent resistance to DIO in A/J mice, we utilized microarray technology to investigate genomic changes in hypothalamic mRNA expression after 2 days and 1 wk of high-fat feeding. Several molecules were identified, including IL-1R1. We thus performed quantitative RT-PCR analysis to assess the expression of not only IL-1R1 and IL-1R2, but also the ligand itself, IL-1{beta}. We observed a significant decrease in the hypothalamic expression of IL-1{beta} and its receptors after 2 days of high-fat feeding, but expression levels returned to levels comparable to those of low-fat diet-fed mice after 1 wk. We also confirmed the expression of another molecule revealed by microarray analysis, i.e., PPM1b, which has been implemented in inhibiting the IL-1{beta}-induced TAK1 signaling pathway (16), a stress-activated pathway that has been demonstrated to signal via the mitogen-activated protein kinase kinase 4-c-Jun NH2-terminal kinase (MKK4-JNK) and mitogen-activated protein kinase kinase 6-p38 (MKK6-p38) pathway (46). Again, we found hypothalamic PPM1b expression to be significantly upregulated after 2 days, but not 1 wk, of high-fat feeding. Because IL-1{beta} and its receptors (IL-1R1 and IL-1R2) were downregulated after 2 days of high-fat feeding and PPM1b, a negative regulator of TAK1-mediated IL-1{beta} action, was upregulated after 2 days of high-fat feeding, it seems that an initial response to a high-fat diet is associated with downregulation of the anorexigenic IL-1{beta} pathway and induction of the inhibition of TAK1-mediated IL-1{beta} signaling. The observed effect of high-fat diet-induced suppression of anorexigenic IL-1{beta} action and induction of hyperphagia in A/J mice lasts <1 wk; therefore, it appears that, between 2 days and 1 wk of exposure to a high-fat diet, the expression of other, not yet identified molecules, which can override the IL-1{beta} system, may be altered and, thus, result in reduction of caloric intake to baseline levels and normalization of the IL-1{beta} pathway. Initial elevation of leptin levels in A/J mice is consistent with the observed initial downregulation of the IL-1{beta} pathway (9, 14, 20). It would be interesting to see whether the initial downregulation of the IL-1{beta} pathway in response to high-fat feeding is persistent in DIO-prone C57Bl/6J mice for longer periods of time and, more importantly, whether molecules known to act upstream of the IL-1 pathway, namely, IL-1Ra, are differentially regulated compared with A/J mice under the same conditions. Under pathological conditions, excess IL-1 signaling has been demonstrated to have potent effects in causing anorexia and fever, acting at the level of the hypothalamus (26, 29), but studies utilizing IL-1–/– mice do not reveal any apparent abnormalities under physiological conditions (26), indicating that lack of IL-1 signaling may be dispensable in the maintenance of body weight. Nonetheless, the exact role of the IL-1 system in the development of DIO resistance in A/J mice needs to be studied further, because it remains possible that, in addition to upregulation of UCP2, other molecules may also contribute to keeping body weight stable, despite excess caloric intake after 2 days of high-fat feeding.

Additional candidate molecules proposed, on the basis of our microarray analysis results, include the following. Ugt1a6 is a member of the UDP-glucuronosyltransferase family of membrane-bound proteins found in the endoplasmic reticulum. They are widely responsible for the glucuronidation of numerous endobiotic and xenobiotic substrates, resulting in increased water solubility and ultimate renal and biliary excretion (28). Asb5 is one of many SOCS-box-containing proteins that have been implemented in the suppression of cytokine signaling (e.g., SOCS-3 inhibition of leptin signaling) (2, 4, 23). Asb5 has not been well characterized, having been discussed in only one study proposing its role in arteriogenesis (4). Because both of these molecules were downregulated after 2 days of high-fat feeding but returned to normal levels at 1 wk, it remains to be studied whether these two molecules may play a role in leptin signaling and/or regulation of caloric intake in response to high-fat feeding of DIO-resistant A/J mice. Future studies validating our microarray data and providing analysis of Ugt1a6 and Asb5 in response to high-fat feeding are needed to further elucidate the potential importance of these changes.

In conclusion, our data are consistent with recent findings indicating that leptin levels are slightly increased in response to high-fat feeding, which, in turn, may activate UCPs (25) within the first 2 days of exposure of A/J mice to high-fat diets. Moreover, we have shown that hypothalamic neuropeptide expression of known modulators of energy homeostasis remains largely unchanged for the duration of the study, indicating that alterations in these hypothalamic neuropeptides, as well as inhibitors of leptin action, do not mediate the early-phase changes in food and caloric intake in DIO-resistant A/J mice. Finally, by utilization of gene array analysis and quantitative RT-PCR, we have exposed the possibility that other peptides possibly acting upstream of the IL-1 system may potentially be involved in mediating early-phase resistance to DIO in A/J mice. These peptides, coupled with increased UCP2 expression in BAT in response to slightly elevated leptin levels and retention of leptin sensitivity, could prove to be the underlying mechanisms driving long-term resistance to DIO in A/J mice. Further studies are needed to confirm and extend our data, focusing on further elucidation of the specific role of PPM1b and the IL-1{beta} pathway in altering food intake in DIO, further investigation into the posttranscriptional regulation of the proteins discussed here, and further classification of the full array of molecules that were revealed by microarray analysis that may alter energy homeostasis.


    FOOTNOTES
 

Address for reprint requests and other correspondence: C. S. Mantzoros, BIDMC, ST 816, 330 Brookline Ave., Boston, MA 02215 (E-mail: cmantzor{at}bidmc.harvard.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Bergin HT, Mizuno T, Taylor J, and Mobbs CV. Resistance to diet-induced obesity is associated with increased proopiomelanocortin mRNA and decreased neuropeptide Y mRNA in the hypothalamus. Brain Res 851: 198–203, 1999.[CrossRef][ISI][Medline]
  2. Bjørbæk C, El-Haschimi K, Frantz JD, and Flier JS. The role of SOCS-3 in leptin signaling and leptin resistance. J Biol Chem 274: 30059–30065, 1999.[Abstract/Free Full Text]
  3. Bjørbæk C, Elmquist J, El-Haschimi K, Kelly J, Ahima RS, Hileman S, and Flier JS. Activation of SOCS-3 messenger ribonucleic acid in the hypothalamus by ciliary neutrophic factor. Endocrinology 140: 2035–2043, 1999.[Abstract/Free Full Text]
  4. Boengler K, Pipp F, Fernandez B, Richter A, Schaper W, and Deindl E. The ankyrin repeat containing SOCS box protein 5: a novel protein associated with arteriogenesis. Biochem Biophys Res Commun 302: 17–22, 2003.[CrossRef][ISI][Medline]
  5. Chemeli RM, Willie JT, Sinton CM, Elmquist JK, Scammell T, Lee C, Richardson JA, Williams SC, Xiong Y, Kisanuki Y, Fitch TE, Nakazato M, Hammer RE, Safer CB, and Yanagisawa M. Narcolepsy in orexin knockout mice: molecular genetics of sleep regulation. Cell 98: 437–451, 1999.[ISI][Medline]
  6. Cheng A, Uetani N, Simoncic PD, Chaubey VP, Lee-Loy A, McGlade CJ, Kennedy BP, and Tremblay ML. Attenuation of leptin action and regulation of obesity by protein tyrosine phosphatase 1B. Dev Cell 2: 497–503, 2002.[ISI][Medline]
  7. Elchebly M, Payette P, Michaliszyn E, Cromlish W, Collins S, Loy AL, Normandin D, Cheng A, Himmus-Hagen J, Chan C, Ramachandran C, Gresser MJ, Tremblay ML, and Kennedy BP. Increased insulin sensitivity and obesity resistance in mice lacking the protein tyrosine phosphatase-1B gene. Science 283: 1544–1548, 1999.[Abstract/Free Full Text]
  8. El-Haschimi K, Pierroz DD, Hileman SM, Bjørbaek C, and Flier JS. Two defects contribute to hypothalamic leptin resistance in mice with diet-induced obesity. J Clin Invest 105: 1827–1832, 2000.[Abstract/Free Full Text]
  9. Faggioni R, Fantuzzi G, Gabay C, Moser A, Dinarello CA, Feingold KR, and Grunfeld C. Leptin deficiency enhances sensitivity to endotoxin-induced lethality. Am J Physiol Regul Integr Comp Physiol 276: R136–R142, 1999.[Abstract/Free Full Text]
  10. Fan W, Boston BA, Keterson RA, Hruby VJ, and Cone RD. Role of melanocortinergic neurons in feeding and the Agouti obesity syndrome. Nature 385: 165–168, 1997.[CrossRef][ISI][Medline]
  11. Fleury C, Neverova M, Collins S, Raimbault S, Champigny O, Levi-Meyrueis C, Bouillaud F, Seldin MF, Surwit RS, Ricquier D, and Warden CH. Uncoupling protein-2: a novel gene linked to obesity and hyperinsulinemia. Nat Genet 15: 269–272, 1997.[ISI][Medline]
  12. Flier J and Maratos-Flier E. Energy homeostasis and body weight. Curr Biol 10: R215–R217, 2000.[CrossRef][ISI][Medline]
  13. Friedman JM and Halaas JL. Leptin and the regulation of body weight. Curr Biol 10: 763–770, 2000.
  14. Gabay C, Dreyer MG, Pellegrinelli N, Chicheportiche R, and Meier CA. Leptin directly induces secretion of interleukin 1 receptor antagonist in human monocytes. J Clin Endocrinol Metab 86: 783–791, 2001.[Abstract/Free Full Text]
  15. Giraudo SQ, Kotz CM, Grace MK, Levine AS, and Billington CJ. Rat hypothalamic NPY mRNA and brown fat uncoupling protein mRNA after high-carbohydrate or high-fat diets. Am J Physiol Regul Integr Comp Physiol 266: R1578–R1583, 1994.[Abstract/Free Full Text]
  16. Hanada M, Ninomiya-Tsuji J, Komaki K, Ohnishi M, Katsura K, Kanamaru R, Matsumoto K, and Tamura S. Regulation of the TAK1 signaling pathway by protein phosphatase 2C. J Biol Chem 276: 5753–5759, 2000.[CrossRef][ISI][Medline]
  17. Hillebrand JJG, de Wied D, and Adan RAH. Neuropeptides, food intake and body weight regulation: a hypothalamic focus. Peptides 23: 2283–2306, 2002.[CrossRef][ISI][Medline]
  18. Hilton DJ. Negative regulators of cytokine signal transduction. Cell Mol Life Sci 55: 1568–1577, 1999.[CrossRef][ISI][Medline]
  19. Hosoi T, Okuma Y, and Nomura Y. Leptin regulates interleukin-1{beta} expression in the brain via the STAT3-independent mechanisms. Brain Res 949: 139–146, 2002.[CrossRef][ISI][Medline]
  20. Juge-Aubry C and Meier CA. Immunomodulatory actions of leptin. Mol Cell Endocrinol 194: 1–7, 2002.[CrossRef][ISI][Medline]
  21. Kalra SP, Dube MG, Pu S, Xu B, Horvath TL, and Kalra PS. Interacting appetite-regulating pathways in the hypothalamic regulation of body weight. Endocr Rev 20: 68–100, 1999.[Abstract/Free Full Text]
  22. Klaman L, Boss O, Peroni O, Kim J, Martino J, Zabalotny J, Moghal N, Lubkin M, Kim Y, Sharpe A, Stricker-Kongrad A, Shulman G, Neel B, and Kahn B. Increased energy expenditure, decreased adiposity, and tissue-specific insulin sensitivity in protein tyrosine phosphatase 1B-deficient mice. Mol Cell Biol 20: 5479–5489, 2000.[Abstract/Free Full Text]
  23. Kokkotou E, Tritos N, Mastaitis J, Slieker L, and Maratos-Flier E. Melanin-concentrating hormone receptor is a target of leptin action in the mouse brain. Endocrinology 142: 680–686, 2001.[Abstract/Free Full Text]
  24. Ludwig DS, Mountjoy KG, Tatro JB, Gillette JA, Frederich RC, Flier JS, and Maratos-Flier E. Melanin-concentrating hormone: a functional melanocortin antagonist in the hypothalamus. Am J Physiol Endocrinol Metab 274: E627–E633, 1998.[Abstract/Free Full Text]
  25. Masaki T, Yoshimichi G, Chiba S, Yasuda T, Noguchi H, Kakuma T, Sakata Y, and Yoshimatsu H. Corticotropin-releasing hormone-mediated pathway of leptin to regulate feeding, adiposity, and uncoupling protein expression in mice. Endocrinology 144: 3547–3554, 2003.[Abstract/Free Full Text]
  26. Matsuki T, Horai R, Sudo K, and Iwakura Y. IL-1 plays an important role in lipid metabolism by regulating insulin levels under physiological conditions. J Exp Med 198: 877–888, 2003.[Abstract/Free Full Text]
  27. Mizuno TM, Kleopoulos SP, Bergen HT, Roberts JL, Priest CA, and Mobbs CV. Hypothalamic pro-opiomelanocortin mRNA is reduced by fasting and [corrected] in ob/ob and db/db mice, but is stimulated by leptin. Diabetes 47: 294–297, 1998. [Corrigenda. Diabetes 47: April 1998, p. 696.][Abstract]
  28. Peters W, te Morsche R, and Roelofs H. Combined polymorphisms in UDP-glucuronosyltransferases 1A1 and 1A6: implications for patients with Gilbert's syndrome. J Hepatol 38: 3–8, 2003.[Medline]
  29. Plata-Salaman C and Ilyin S. Interleukin-1{beta} (IL-1{beta})-induced modulation of the hypothalamus IL-1{beta} system, tumor necrosis factor-{alpha}, and transforming growth factor-1{beta} mRNAs in obese (fa/fa) and lean (Fa/Fa) Zucker rats: implications to IL-1{beta} feedback systems and cytokine-cytokine interactions. J Neurosci Res 49: 541–550, 1997.[CrossRef][ISI][Medline]
  30. Prpic V, Watson PM, Frampton IC, Sabol MA, Jezek GE, and Gettys TW. Differential mechanisms and development of leptin resistance in A/J versus C57BL/6J mice during diet-induced obesity. Endocrinology 144: 1155–1163, 2003.[Abstract/Free Full Text]
  31. Prpic V, Watson PM, Frampton IC, Sabol MA, Jezek GE, and Gettys TW. Adaptive changes in adipocyte gene expression differ in AKR/J and SWR/J mice during diet-induced obesity. J Nutr 132: 3325–3332, 2002.[Abstract/Free Full Text]
  32. Qu D, Ludwig DS, Gammeltoft S, Piper M, Pelleymounter MA, Cullen MJ, Mathes WF, Pryspek R, Kanarek R, and Maratos-Flier E. A role for melanin-concentrating hormone in the central regulation of feeding behaviour. Nature 380: 243–247, 1996.[CrossRef][ISI][Medline]
  33. Rebuffe-Scrive M, Surwit R, Feinglos M, Kuhn C, and Rodin J. Regional fat distribution and metabolism in a new mouse model (C57BL/6J) of non-insulin-dependent diabetes mellitus. Metabolism 42: 1405–1409, 1993.[ISI][Medline]
  34. Rosenbaum M, Leibel RL, and Hirsch J. Obesity. N Engl J Med 337: 396–407, 1997.[Free Full Text]
  35. Sakurai T, Amemiya A, Ishii M, Matsuka I, Chemeli RM, Tanaka H, Williams SC, Richarson JA, Kozlowski GP, and Wilson S. Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior. Cell 92: 573–585, 1998.[ISI][Medline]
  36. Shimada M, Tritos NA, Lowell BB, Flier JS, and Maratos-Flier E. Mice lacking melanin-concentrating hormone are hypophagic and lean. Nature 396: 670–674, 1998.[CrossRef][ISI][Medline]
  37. Starr R and Hilton DJ. Negative regulation of the JAK/STAT pathway. Bioessays 21: 47–52, 1999.[CrossRef][ISI][Medline]
  38. Surwit RS, Feinglos MN, Rodin J, Sutherland A, Petro AE, Opara EC, Kuhn CM, and Rebuffe-Scrive M. Differential effects of fat and sucrose on the development of obesity and diabetes in C57BL/6J and A/J mice. Metabolism 44: 644–651, 1995.
  39. Surwit RS, Petro AE, Parekh P, and Collins S. Low plasma leptin in response to dietary fat in diabetes- and obesity-prone mice. Diabetes 46: 1516–1520, 1997.[Abstract]
  40. Surwit RS, Wang S, Petro AE, Sanchis D, Raimbault S, Ricquier D, and Collins S. Diet-induced changes in uncoupling proteins in obesity-prone and obesity-resistant strains of mice. Proc Natl Acad Sci USA 95: 4061–4065, 1998.[Abstract/Free Full Text]
  41. Taheri S, Ward H, Ghatei M, and Bloom S. Role of orexins in sleep and arousal mechanisms. Lancet 335: 847, 2000.
  42. Ukkola O and Santaniemi M. Protein tyrosine phosphatase 1B: a new target for the treatment of obesity and associated co-morbidities. J Intern Med 251: 467–475, 2002.[CrossRef][ISI][Medline]
  43. Watson PM, Commins SP, Beiler RJ, Hatcher HC, and Gettys TW. Differential regulation of leptin expression and function in A/J vs. C57BL/6J mice during diet-induced obesity. Am J Physiol Endocrinol Metab 279: E356–E365, 2000.[Abstract/Free Full Text]
  44. West DB, Waguespack J, and McCollister S. Dietary obesity in the mouse: interaction of strain with diet composition. Am J Physiol Regul Integr Comp Physiol 268: R658–R665, 1995.[Abstract/Free Full Text]
  45. Wlk M, Wang CC, Venihaki M, Liu J, Zhao D, Anton PM, Mykoniatis A, Pan A, Zacks J, Karalis K, and Pothoulakis C. Corticotropin-releasing hormone antagonists possess anti-inflammatory effects in mouse ileum. Gastroenterology 123: 505–515, 2002.[CrossRef][ISI][Medline]
  46. Yamaguchi K, Shirakabe K, Shibuya H, Irie K, Oishi I, Ueno N, Taniguchi T, Nishida E, and Matsumoto K. Identification of a member of the MAPKKK family as a potential mediator of TGF-{beta} signal transduction. Science 270: 2008–2011, 1995.[Abstract]
  47. Zabolotny JM, Bence-Hanulec KK, Stricker-Krongrad A, Haj F, Wang Y, Minokoshi Y, Kim YB, Elmquist JK, Tartaglia LA, Kahn BB, and Neel BG. PTP1B regulates leptin signal transduction in vivo. Dev Cell 2: 489–495, 2002.[ISI][Medline]
  48. Ziotopoulou M, Mantzoros CS, Hileman SM, and Flier JS. Differential expression of hypothalamic neuropeptides in the early phase of diet-induced obesity in mice. Am J Physiol Endocrinol Metab 279: E838–E845, 2000.[Abstract/Free Full Text]




This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Search for citing articles in:
ISI Web of Science (2)
Google Scholar
Articles by Bullen, J. W.
Articles by Mantzoros, C. S.
Articles citing this Article
PubMed
PubMed Citation
Articles by Bullen, J. W., Jr
Articles by Mantzoros, C. S.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2004 by the American Physiological Society.