Hyperleptinemia in Ay/a mice upregulates arcuate cocaine- and amphetamine-regulated transcript expression

Yoshio Tsuruta, Hironobu Yoshimatsu, Shuji Hidaka, Seiya Kondou, Kenjiro Okamoto, and Toshiie Sakata

Department of Internal Medicine I, School of Medicine, Oita Medical University, Oita 879-5593, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The effects of leptin on cocaine- and amphetamine-regulated transcript (CART) and agouti-related protein (AGRP) expression in the hypothalamic arcuate nucleus of obese Ay/a mice were investigated. CART mRNA expression was upregulated by 41% and AGRP mRNA downregulated by 78% in hyperleptinemic Ay/a mice relative to levels in lean a/a mice. The mRNA expression of these neuropeptides in either young nonobese Ay/a mice or rats treated with SHU-9119, a synthetic melanocortin-4 receptor (MC4R) antagonist, did not differ significantly from that in the corresponding controls. After a 72-h fast, which decreased the concentration of serum leptin, CART and AGRP mRNA expression decreased and increased, respectively, in Ay/a mice. The expression levels of these neuropeptides in leptin-deficient Ay/a ob/ob double mutants were comparable to those in a/a ob/ob mice. Leptin thus modulates both CART and AGRP mRNA expression in obese Ay/a mice, whereas leptin signals are blocked at the MCR4R level. Taken together, the present findings indicate that differential expression of these neuropeptides in Ay/a and ob/ob mice results in dissimilar progression toward obesity.

leptin; lethal yellow mice; agouti-related protein; arcuate nucleus


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

LEPTIN HAS BEEN SHOWN TO INDUCE anorectic effects in rodents, at least in part, by signaling through the melanocortin-4 receptor (MC4R) (36). Agouti protein has a potent antagonistic effect on hypothalamic MC4R (7, 30), and rodents become hyperphagic when signaling is interrupted (19, 31). Lethal yellow (Ay/a) mice display ectopic overexpression of agouti protein in the brain, which results in defective proopiomelanocortin (POMC)/MC4R signaling and, upon maturation, obesity (7, 16, 30, 44). Because these obese mice are both hyperleptinemic and insensitive to exogenous leptin treatment (16, 44), they are thought to be in a "leptin-resistant state." However, it has been reported that the major effects of POMC- and leptin-induced signals on body weight are independent and additive in Ay/a ob/ob doubly mutant mice (3). In addition, full leptin responsiveness is restored in Ay/a ob/ob mice (3). These findings indicate that defective signaling through MC4R does not necessarily impair leptin action completely. It is quite likely that leptin continues to act, at least in part, on some hypothalamic neuropeptides in Ay/a mice.

Cocaine- and amphetamine-regulated transcript (CART) was originally identified as a hypothalamic neuropeptide upregulated by cocaine and amphetamine treatment (6). Central administration of CART induces c-fos expression in several hypothalamic nuclei related with feeding control (11, 42). Recombinant CART fragments decreased food intake in rodents (1, 29, 42), whereas anti-CART antibodies increased it (24). Asnicar et al. (2) have recently demonstrated that absence of CART results in obesity in mice fed a high-calorie diet. Moreover, the solution structure of the CART peptide reveals that a common protein fold displays a novel functionality: affecting food intake (27). In contrast, infusion of agouti-related protein (AGRP) into the cerebroventricle accelerates the rate of rodent feeding (26, 34, 38), because AGRP is competitively antagonistic against alpha -melanocyte-stimulating hormone (alpha -MSH), one of the end products of POMC, at levels of MC3R and MC4R (29, 44). Overexpression of AGRP in transgenic mice (31) produces an obese phenotype similar to that of the MC4R knockout (19) or Ay/a mice (7, 30). Hypothalamic AGRP has also been found to downregulate the hypothalamo-pituitary-thyroid (HPT) axis through leptin-mediated effects on the HPT system (23). POMC and CART were subsequently found to colocalize in discrete ARC neurons (11). Neuropeptide Y (NPY) and AGRP co-localize in the hypothalamic arcuate nucleus (ARC) (15). Most POMC/CART and NPY/AGRP neurons express the leptin receptor in its long form (15, 39), through which leptin conveys different messages to each type of neuron; leptin upregulates CART mRNA expression (12, 24) and downregulates AGRP mRNA (10, 37, 44). Both leptin-deficient ob/ob mice and leptin receptor-deficient Zucker fa/fa rats have decreased CART mRNA levels in the ARC (24). However, AGRP mRNA levels were increased in the nuclei of neurons in ob/ob and leptin receptor-deficient db/db mice (9, 37). CART and AGRP, hypothalamic neuropeptides whose expression is modulated directly by leptin, are thus essential for controlling energy homeostasis.

The aims of the present study are twofold: first, to investigate whether leptin differentially affects CART and AGRP mRNA expression in Ay/a mice, and second, to investigate whether, by production of genetically engineered Ay/a ob/ob doubly mutant mice, distinct phenotypes in genetically obese mice may be attributable to different effects of leptin on CART and AGRP in the course of progression toward obesity.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Animals. Male Wistar King A (WKA) rats at 12 wk, male lethal yellow (Ay/a) mice on the C57BL/6J background, their lean (a/a) littermates at 4-5 wk ("young") and 16-18 wk ("mature"), male ob/ob, and doubly mutant Ay/a ob/ob mice at 16-18 wk (mature) were used. Five mice were used in each group. All animals were housed individually in a room illuminated daily from 0700 to 1900 (a 12:12-h light-dark cycle) at a temperature of 21 ± 1°C and 55 ± 5% humidity. All animals were allowed free access to tap water and standard mouse chow (CE-2, CLEA Japan, Tokyo, Japan) unless otherwise noted. All experiments were in accordance with the Oita Medical University Animal Guidelines, based on the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Production of doubly mutant Ay/a ob/ob mice and their genotyping. The procedures used in establishing this model have been described elsewhere (4). Briefly, C57BL/6J mice with either the Ay/a or ob/+ genotypes (Jackson Labs, Bar Harbor, ME) were crossbred to create lethal yellow obese breeders (Ay/a ob/+). The animals were bred to create doubly mutant Ay/a ob/ob mice, which were identifiable by their coat color and obese phenotype. Mouse genotyping was performed using polymerase chain reaction (PCR) followed by enzymatic cleavage to identify the presence or absence of the leptin mutation (4). Genomic DNA was prepared from whole blood, after which InstaGene Matrix (Bio-Rad Laboratories, Hercules, CA) was used for DNA extraction. Twelve microliters of template aliquots were subsequently subjected to PCR amplification. The PCR primers used were 5'-CTG GTT CTT CAC GGA TAT CAT TG-3' and 5'-AGG GAG CAG CTC TTG GAG AA-3' (4). The amplification reaction was carried out in 30 µl for 35 cycles at 95, 53, and 72°C at 30 s per cycle, following which a discrete 472-bp product was obtained. A 20-µl aliquot of the product was then digested with DdeI (Toyobo, Osaka, Japan) and fractionated on a native 2.5% agarose gel. Staining the gel with ethidium bromide allowed the products to be visualized under ultraviolet transillumination.

Surgical procedures for placing a chronically indwelling catheter into the brain. Male WKA rats were given intraperitoneal pentobarbital sodium anesthesia (45 mg/kg) and placed in a stereotaxic apparatus (Narishige, Tokyo, Japan). Rats were given a chronic implantation into the third cerebroventricle (i3vt) with a stainless steel guide cannula (23 gauge) >= 7 days before the onset of infusion. A stainless steel wire stylet (29 gauge) was inserted into the guide cannula to prevent both leakage of the cerebrospinal fluid and obstruction of the cannula. Details of the surgical procedures have been described elsewhere (13).

Surgical procedures for blood sampling. Under ether anesthesia, each rat and mouse received a Silastic catheter implant (No. 00, Shinnetsu, Tokyo, Japan) 7 days before serum sample collection. The catheter was inserted through the right jugular vein with the inner end fixed at the entrance to the right atrium. A sampling tube was attached to a 21-gauge multisampling needle (Terumo, Tokyo, Japan) to prevent air from being sucked into the system. Details of chronic catheter insertion and blood sampling procedures have been described elsewhere (35).

Collection of blood samples and brain extirpation. Blood samples were taken via the implanted catheter immediately before brain excision to measure blood- and adipocyte-borne humoral factors. Serum samples were immediately frozen at -30°C until used in subsequent assays. Each brain was rapidly excised at 1000 and chilled on an ice plate according to previously described methods (14). Chilled brains of mice were sectioned at -1.00 and -2.85 mm relative to the interaural line according to the mouse brain atlas (33) and chilled rat brains at -4.48 and -6.88 mm relative to the interaural line according to the rat atlas (32). Blocked sections were frozen in liquid nitrogen and stored at -80°C before ARC removal. The samples were prepared by slicing serially on a frozen stage with a sliding microtome, after which the ARC was punched out from frozen brain slices. Total RNA was immediately extracted from the tissues.

Measurement of food consumption, body weight, circulating substances, and neuropeptide mRNAs in WKA rats with SHU-9119. On the day of infusion, a test solution of SHU-9119 (SHU) (Phoenix Pharmaceuticals, Mountain View, CA) in saline was freshly prepared at a concentration of 1.0 nmol/10.0 µl. The pH of SHU and saline control test solutions was adjusted to 6.8-7.2. After being matched according to body weight and food intake during the adaptation period, 15 WKA rats were assigned to one of the following three groups: a group treated with SHU and fed ad libitum (SHU ad lib), a group treated with SHU and pair-fed with the same volume of food intake as the saline controls (SHU pair-fed), and a group treated with saline and fed ad libitum (SAL ad lib). On the day of infusion, the insertion cannula was replaced with a 29-gauge injection cannula. Both SHU-treated groups were infused with 1.0 nmol/10.0 µl SHU at a rate of 1.0 µl/min for 10 min, whereas the SAL group was infused with the same volume of saline through the i3vt cannula at 1000. The infusion and ingestion procedures were performed without restraint or anesthesia (41). The details of the infusion study and food intake measurement have been described elsewhere (13, 35). After i3vt infusion of the test solutions, cumulative food intake and body weight for each rat were measured over a 2-h period in a testing chamber (30 × 25 × 25 cm) equipped with an eatometer, drinkometer, and ambulation-detecting photobeam (35). A bolus infusion of 1.0 nmol/rat SHU has been previously ascertained to reduce food intake significantly (36). Immediately after these measurements, blood samples were taken for measurement of serum substances. After the sampling, the brains were removed for measurement of neuropeptide mRNAs.

Assays of blood- and adipocyte-borne humoral factors. Serum glucose, insulin, and leptin concentrations were measured using a commercially available kit for glucose measurement (Merckauto Glucose, Kanto Chemical, Tokyo, Japan), a rat insulin 125I radioimmunoassay (RIA) kit (Amersham, Little Chalfont, UK), and a rat leptin RIA kit (Linco, St. Louis, MO), respectively. Serum corticosterone concentration was measured using a rat corticosterone 125I RIA kit (Amersham).

RNA extraction and Northern blot analysis. Total RNA was extracted using the standard acid guanidinium-phenol-chloroform method (18). Details of the Northern blot analysis procedure have been described elsewhere (18). Briefly, cDNA probes for mouse CART and AGRP were prepared by RT-PCR by use of the following primers: CART-sense, 5'-AAG TCC AGC ACC ATG GAG AG-3'; CART-antisense, 5'-TCT TCA TTG TGA CCC GCC AC-3'; AGRP-sense, 5'-CAT GCT GAC TGC AAT GTT GC-3'; AGRP-antisense, 5'-CTA GGT GCG ACT ACA GAG G-3'. The identity of those products with the appropriate size was confirmed by mapping with multiple restriction endonucleases and sequencing. The hybridization signals were analyzed with a BIO-image analyzer BAS 2000 (Fuji, Tokyo, Japan). Band intensities obtained by Northern blots were normalized to those of 18S ribosomal RNAs (rRNAs). Western blot analysis was not performed to assess CART or AGRP protein levels, because the total amount of protein extracted from the ARC punched out of frozen brain slices was too small.

Statistical analysis. Each value was expressed as the mean ± SE. Statistical significance was assessed by two-way analysis of variance (ANOVA) with repeated measurements. Differences were considered significant at P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Differences in body weight, circulating substances, and neuropeptide mRNA levels between young and mature Ay/a mice. The mature obese lethal yellow (Ay/a) ad libitum-fed group showed more prominent body weight gain (P < 0.05), hyperinsulinemia (P < 0.01), and hyperleptinemia (P < 0.001) than the lean littermate control (a/a) group. However, serum corticosterone levels were comparable between the groups (Table 1). CART mRNA expression was 41% higher in the mature obese Ay/a group [0.156 ± 0.014, mRNA/rRNA arbitrary units (a.u.)] compared with the lean control group (0.111 ± 0.013, mRNA/rRNA a.u.; P < 0.01) (Fig. 1A). AGRP mRNA expression, in contrast, was 78% lower in the obese Ay/a group (0.018 ± 0.004, mRNA/rRNA a.u.) relative to the lean control group (0.083 ± 0.007, mRNA/rRNA a.u.; P < 0.001) (Fig. 1B). Notably, there was no significant difference in body weight (15.2 ± 0.7 g in Ay/a vs. 15.7 ± 0.7 g in a/a), serum glucose concentration (209 ± 23 vs. 177 ± 17 mg/dl), insulin concentration (0.9 ± 0.1 vs. 0.9 ± 0.1 ng/ml), or leptin concentration (2.1 ± 0.7 vs. 2.0 ± 0.5 ng/ml) between the young Ay/a and a/a groups. With regard to these parameters, there was no significant difference in CART or AGRP gene expression between young Ay/a mice and their controls (Fig. 1, A and B).

                              
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Table 1.   Comparison of body weight and circulating substances among lean control (a/a+/+), lethal yellow (Ay/a+/+), leptin-deficient (a/a ob/ob), and doubly mutant (Ay/a ob/ob) mice



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Fig. 1.   Difference in mRNA expression of cocaine- and amphetamine-regulated transcript (CART; A) and agouti-related protein (AGRP; B) between mature (16-18 wk old) and young (4-5 wk old) lethal yellow (Ay/a) and lean littermate control (a/a) male mice. In this figure, as in Figs. 2-4, A and B, top: neuropeptide gene expression and the corresponding 18S rRNA, and A and B; bottom: relative changes in neuropeptide mRNA levels in the arcuate nucleus. Total RNA (5 µg/lane) was analyzed; each lane contains RNA from 1 mouse. In all figures, each bar represents the mean ± SE (n = 5 per group). * P < 0.01, ** P < 0.001 vs. corresponding a/a controls.

Effects of SHU-9119 on body weight, circulating substances, and neuropeptide mRNAs in WKA rats. The SHU ad lib group ate more voraciously than the SAL ad lib controls over the course of the 2-h feeding period, as measured by cumulative food intake (SHU ad lib, 2.4 ± 0.5 g; SAL ad lib, 0.2 ± 0.1 g; P < 0.001). However, no differences were found among the SHU ad lib, SHU pair-fed, and SAL ad lib groups in body weight (415 ± 9, 413 ± 13, and 428 ± 6 g, respectively), serum glucose (132 ± 4, 126 ± 2, and 125 ± 3 mg/dl), or leptin concentration (3.4 ± 0.8, 5.1 ± 0.7, and 4.7 ± 0.8 ng/ml). In contrast, serum insulin levels in the SHU ad lib group (7.1 ± 0.6 ng/ml) were higher than those in the SHU pair-fed group (4.5 ± 0.5 ng/ml, P < 0.01), although neither value differed significantly from that in the SAL ad lib group (5.6 ± 0.3 ng/ml). As shown in Fig. 2, SHU did not affect mRNA expression of CART or AGRP in either the SHU ad lib or the SHU pair-fed group.


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Fig. 2.   Effects of SHU-9119 infusion by chronic implantation into 3rd cerebroventricle on CART (A) and AGRP (B) mRNA expression in Wistar King A rats. Total RNA (10 µg/lane) was analyzed; each lane contains RNA from 1 rat (n = 5 per group). SAL, control group infused with saline; SHU, experimental group with SHU-9119; ad lib, ad libitum fed; pair-fed, fed with the same volume of food as the SHU ad lib group.

Effects of 72-h fast on body weight, circulating substances, and neuropeptide mRNA levels in Ay/a mice. Mature Ay/a mice fasted for 72 h displayed prominent weight loss (P < 0.01), hypoglycemia (P < 0.001), hypoinsulinemia (P < 0.001), and hypoleptinemia (P < 0.01) compared with corresponding Ay/a mice fed ad libitum (Table 1). The 72-h fasting, however, did not affect serum corticosterone concentration in either group (Table 1). CART mRNA was downregulated in the 72-h fasted Ay/a mice relative to corresponding mice fed ad libitum (P < 0.01) (Fig. 3A). In contrast, fasted Ay/a mice upregulated AGRP mRNA relative to mice fed ad libitum (P < 0.01) (Fig. 3B). Furthermore, fasting for 72 h restored the AGRP and CART mRNAs in Ay/a mice to almost exactly the wild-type expression levels.


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Fig. 3.   Effects of 72-h fasting on CART (A) and AGRP (B) mRNA expression in mature Ay/a mice. Total RNA (5 µg/lane) was analyzed; each lane contains RNA from 1 mouse. Fed, ad lib group. Fasted, fasted for 72 h; n = 5 per group. * P < 0.01 vs. ad lib mice.

Effects of additional ob gene mutation on body weight, circulating substances, and neuropeptide mRNA levels in Ay/a mice. The a/a ob/ob mice showed increases in all parameters measured, including body weight (P < 0.001), serum glucose concentration (P < 0.05), serum insulin concentration (P < 0.01), and serum corticosterone concentration (P < 0.01), relative to a/a +/+ controls (Table 1). They also showed greater weight gain (P < 0.01) and higher serum insulin concentration (P < 0.001) than Ay/a +/+ mice (Table 1). The doubly mutant Ay/a ob/ob mice also displayed weight gain (P < 0.001 vs. a/a +/+; P < 0.01 vs. Ay/a +/+), hyperglycemia (P < 0.001 vs. a/a +/+; P < 0.01 vs. Ay/a +/+), hyperinsulinemia (P < 0.01 vs. a/a +/+; P < 0.001 vs. Ay/a +/+) and hypercorticosteronemia (P < 0.01 vs. a/a +/+; P < 0.01 vs. Ay/a +/+) relative to their controls (Table. 1). Serum leptin was not detectable in either a/a ob/ob or Ay/a ob/ob mice. In addition, there was no significant difference between a/a ob/ob and Ay/a ob/ob mice in the various parameters shown in Table 1. As shown in Fig. 4A, CART mRNA expression was downregulated in a/a ob/ob mice relative to that in a/a +/+ mice (P < 0.01) and Ay/a +/+ mice (P < 0.001). A similar decrease in CART mRNA expression was observed in Ay/a ob/ob mice relative to that in a/a +/+ (P < 0.05) and Ay/a +/+ mice (P < 0.001). The reduced CART mRNA expression levels in a/a ob/ob and Ay/a ob/ob mice were comparable in value (Fig. 4A). On the other hand, AGRP mRNA expression was upregulated to a similar extent in a/a ob/ob and Ay/a ob/ob mice, and both values were higher than those in a/a +/+ (P < 0.001) and Ay/a +/+ groups (P < 0.0001) (Fig. 4B).


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Fig. 4.   Effects of single and double (i.e., combined) mutations in the Ay/a and ob/ob genes on CART (A) and AGRP (B) mRNA expression in the arcuate nucleus. Total RNA (5 µg/lane) was analyzed; each lane contains RNA from 1 mouse (n = 5 per group). * P < 0.05, ** P < 0.01, *** P < 0.001 vs. a/a +/+ group. dagger  P < 0.001, dagger dagger P < 0.0001 vs. Ay/a +/+ group.


    DISCUSSION
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ABSTRACT
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The present study demonstrates that mRNA expression of CART and AGRP was, respectively, upregulated and downregulated in the ARC of mature Ay/a mice. The present results are consistent with previous findings (9) that AGRP mRNA expression was downregulated in these mice. Furthermore, the results presented here show that mature Ay/a mice are hyperinsulinemic and hyperleptinemic. This is also in line with previous findings (3, 7, 16, 30), although these characteristics were milder in Ay/a mice than in ob/ob mice (with the exception of hyperleptinemia). In the present study, we were not able to assess CART or AGRP protein levels and/or activity, because the amount of total protein recovered was too small. However, previous reports have shown that CART and AGRP function is reflected in mRNA expression levels (21, 25). Taken together, the previous and present results provide several insights into how anorectigenic CART and orexigenic AGRP are regulated in the Ay/a ARC.

One possible explanation is that agouti protein and/or blockade of MC4R modulates mRNA expression of both CART and AGRP in the mature Ay/a ARC. Unlike mature Ay/a mice, the young nonobese Ay/a mice studied here showed normal CART and AGRP mRNA expression. In addition, i3vt infusion of SHU-9119, a synthetic antagonist of MC4R (36), did not change the mRNA expression of these neuropeptides in wild WKA rats, leaving serum leptin concentrations unaffected while increasing food consumption. These findings suggest that CART and AGRP mRNA expression is not affected by either acute or chronic blockade of MC4R signaling. This supports previous reports that anorectic CART acts independently of the melanocortin-signaling pathway (10) as well as other studies, in which leptin inhibited AGRP mRNA expression in KKAy mice independently of MC4R signaling (9). Thus it seems unlikely that agouti protein overexpression and/or MC4R signal interruption affect CART and/or AGRP mRNA expression. Rather, the present and previous findings together suggest that CART and/or AGRP mRNA expression is independent of agouti protein overexpression and/or MC4R signal interruption.

An alternative explanation is that CART and AGRP transcripts may be affected by hyperinsulinemia or hyperleptinemia in mature Ay/a mice, since serum concentrations of insulin and leptin do not increase in young preobese mice in this study. To confirm the effects of these factors on the transcripts, we deprived mature Ay/a mice of food to decrease circulating leptin levels due to loss of body weight. In marked contrast to the ad libitum-fed Ay/a mice, 72-h fasting normalized the expression of both transcripts to wild-type levels while lowering serum leptin concentrations as well. It has been reported elsewhere that leptin normally increases CART mRNA expression (24) and decreases AGRP mRNA expression (9, 37), whereas expression levels of both neuropeptides in leptin-deficient animals are opposite those in leptin-treated rodents (17, 43). These findings support our observations that CART mRNA expression decreased whereas AGRP mRNA expression increased in a/a ob/ob mice relative to a/a mice. Although not demonstrated conclusively by the postfasting data, hyperleptinemia most likely produces the observed changes in neuropeptide mRNA expression in mature Ay/a mice. Leptin is thus likely to be a key modulator of expression for both neuropeptides in Ay/a and ob/ob mice.

To test this hypothesis and confirm the essential role of leptin in the results presented here, we performed an additional study using MC4R-insensitive and leptin-deficient Ay/a ob/ob doubly mutant mice. Both CART and AGRP mRNA expression levels in the doubly mutant Ay/a ob/ob ARC were opposite those in the Ay/a +/+ ARC. The neuropeptidergic phenotypes in Ay/a ob/ob mice were not significantly different from those in a/a ob/ob mice. In addition, fasting has been reported to have no effect on AGRP mRNA expression in ob/ob mice (44), although leptin supplementation normalizes CART and AGRP gene expression in the mice (9, 24, 44). In the present study, therefore, the doubly mutant mice were not subjected to either leptin treatment or fasting. The remaining potential factor, namely hyperinsulinemia, is less likely to be a key modulator. The rationale behind this relies on the finding that all parameters used were elevated in Ay/a ++, a/a ob/ob, and Ay/a ob/ob mice, whereas CART and AGRP gene expression differed among the groups. The variation in CART and AGRP gene expression seen in Ay/a+/+, a/a ob/ob, and Ay/a ob/ob mice, despite the fact that each group serves as an animal model for obesity, reinforces our theory that obesity per se is not a key modulator for alteration of neuropeptide mRNA expression. Another well known and important modulator affecting obesity development is corticosterone (20, 40). Exogenous leptin is known to manifest its full potent effects on adrenalectomized Ay/a ob/ob mice supplemented regularly with corticosterone (3). Consistent with previous reports (3, 19), our present study proves that serum corticosterone concentration in mature Ay/a mice remains within its normal range. Taking this evidence as a whole, it appears unlikely that corticosterone is a key modulator of these neuropeptides. The present results thus imply a direct association between hyperleptinemia and both elevated CART mRNA and lowered AGRP mRNA expression in the Ay/a ARC independent of the lethal yellow mutation (although again, the present study did not conduct leptin treatment or fasting with the doubly mutant mice).

Current evidence indicates that CART and AGRP colocalize with POMC (12) and neuropeptide Y (NPY) (15), respectively, in subsets of ARC neurons. All of these neuropeptides are regulated directly by leptin (9, 12, 15, 24, 37, 39, 44). However, previous studies have shown that POMC and NPY mRNA levels in the ARC of Ay/a mice do not differ significantly from those in controls (22), whereas the present study shows that hyperleptinemia in mature Ay/a mice results in CART mRNA upregulation and AGRP mRNA downregulation in the ARC. The various neuropeptides thus do not appear to be regulated by leptin in the same way, despite the fact that they have been found to co-localize within the same neurons. In other words, CART and AGRP in Ay/a mice may be regulated downstream of leptin signal messages, whereas body weight and food consumption of these mice may be regulated upstream and are therefore less leptin sensitive (16, 44). These findings provide a key insight into understanding leptin resistance in obesity. Leptin resistance in the regulation of food intake and body weight is not necessarily a result of faulty leptin action on each hypothalamic neuropeptide. Rather, it may result from the combined effects of leptin action on neuropeptides that are sensitive or insensitive to it in the obese state.

It has been well established that Ay/a mice are moderately hyperphagic with mature-onset obesity (5, 45). On the other hand, ob/ob mice are voraciously hyperphagic with early-onset obesity (17, 43). In view of these findings, the difference in phenotype between these mice is likely due to dissimilar effects of leptin signaling on CART and AGRP gene regulation. CART and AGRP have been shown to modulate leptin signals positively and negatively, respectively, and to regulate sympathetic nerve activity (9, 24, 44). Neuroanatomic studies have revealed that CART neurons project directly toward thoracic sympathetic preganglionic neurons that innervate both brown adipose tissue and the heart (8, 12). CART and AGRP neurons in Ay/a mice thus modulate not only energy expenditure but also cardiovascular functions through such leptin-controlled efferent sympathetic pathways. Previous studies focusing on AGRP suggested that AGRP acts through an MC4R-independent pathway (28). The difference in AGRP expression in Ay/a and ob/ob mice reflects distinct progressions toward obesity in each mouse model, which act through an AGRP-dependent but melanocortin-independent pathway not yet determined. With the CART effects described here taken into account, the results can be understood more clearly. The present and previous findings thus suggest that the discrepancy in obesity phenotype between Ay/a and ob/ob mice discussed here arises from differences in leptin manipulation of CART and AGRP gene expression. In light of all this, it seems probable that there may be leptin-sensitive and leptin-insensitive neurons, which ultimately manifest themselves in a distinct progression toward obesity.


    ACKNOWLEDGEMENTS

We thank Dr. David Knight, Department of Cellular Biology and Anatomy, Louisiana State University Health Science Center, and Dr. Tetsuya Kakuma, Department of Internal Medicine I, School of Medicine, Oita Medical University, for help in preparation of the manuscript. We also thank Dr. Reiko Hanada, Department of Internal Medicine I, School of Medicine, Oita Medical University, for technical assistance.


    FOOTNOTES

This work was supported partly by Grant-in-Aid 10045072 (T. Sakata) from the Japanese Ministry of Education, Science and Culture and by research grants from the Japanese Fisheries Agency for Research into Efficient Exploitation of Marine Products for Promotion of Health, 1998-1999 (T. Sakata).

Address for reprint requests and other correspondence: T. Sakata, Dept. of Internal Medicine I, School of Medicine, Oita Medical Univ., 1-1 Idaigaoka, Hasama, Oita, 879-5593 Japan (E-mail: sakata{at}oita-med.ac.jp).

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

10.1152/ajpendo.00292.2001

Received 12 July 2001; accepted in final form 5 December 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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