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Endocrinology, doi:10.1210/en.2004-0263
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Endocrinology Vol. 145, No. 8 3881-3891
Copyright © 2004 by The Endocrine Society

Metabolic Effects of Transgenic Melanocyte-Stimulating Hormone Overexpression in Lean and Obese Mice

Eriika Savontaus, Tracy L. Breen, Andrea Kim, Lucy M. Yang, Streamson C. Chua, Jr. and Sharon L. Wardlaw

Departments of Medicine and Pediatrics (T.L.B., A.K., L.M.Y., S.C.C., S.L.W.), Columbia University College of Physicians and Surgeons, New York, New York 10032; and Department of Pharmacology and Clinical Pharmacology (E.S.), University of Turku, Turku 20520, Finland

Address all correspondence and requests for reprints to: Sharon L. Wardlaw, M.D., Department of Medicine, Columbia University College of Physicians and Surgeons, 630 West 168th Street, New York, New York 10032. E-mail: sw22{at}columbia.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The proopiomelanocortin-derived peptide, {alpha}-MSH, inhibits feeding via melanocortin receptors in the hypothalamus and genetic defects inactivating the melanocortin system have been shown to lead to obesity in experimental animals and humans. To determine whether long-term melanocortinergic activation has significant effects on body weight and composition and insulin sensitivity, transgenic mice overexpressing N-terminal proopiomelanocortin, including {alpha}- and {gamma}3-MSH, under the control of the cytomegalovirus-promoter were generated. The transgene was expressed in multiple tissues including the hypothalamus, in which both {alpha}-MSH and {gamma}3-MSH levels were increased approximately 2-fold, compared with wild-type controls. Transgene homozygous mice were also crossed with obese leptin receptor-deficient db3J and obese yellow Ay mice. MSH overexpression led to uniform, dose- dependent darkening of coat color. MSH overexpression reduced weight gain and adiposity and improved glucose tolerance in lean male mice. In female transgenic mice, there was no significant effect on body weight, but there was a significant decrease in insulin levels. Obesity was attenuated in obese db3J/db3J male and female mice, but there was no improvement in glucose metabolism. In contrast, the MSH transgene improved glucose tolerance in male Ay mice. These results support the hypothesis that long-term melanocortinergic activation could serve as a potential strategy for anti-obesity and/or antidiabetic therapy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE HYPOTHALAMIC MELANOCORTIN neuropeptide system plays a key role in the regulation of body energy homeostasis (1, 2, 3). Proopiomelanocortin (POMC) derived {alpha}-MSH inhibits and the endogenous antagonist agouti-related peptide (AGRP) stimulates feeding via G protein-coupled melanocortin receptors (MC-Rs). Mice with targeted disruption of the POMC gene are obese (4). Obesity also results from global overexpression of AGRP (5) or agouti, a protein normally expressed only in skin in which it affects pigmentation but in obese yellow (Ay) mice agouti is expressed ubiquitously, particularly in the brain (6, 7). In humans, a number of different mutations leading to POMC deficiency have been described that cause severe infant-onset obesity (8, 9).

The MC4- and MC3-Rs mediate the melanocortin effects on energy balance. These receptors are highly expressed in hypothalamic nuclei known to regulate energy homeostasis; MC4-R in the paraventricular nucleus, which contains POMC and AGRP fiber tracts (6) and MC3-R in the arcuate nucleus, in which it is hypothesized to mediate interactions between POMC and AGRP neurons (10, 11). The MC3-R is also expressed in peripheral tissues such as adipose tissue, in which it could be important in regulating energy storage and utilization (12). Targeted disruptions of the MC4- or MC3-R genes result in obesity syndromes with different characteristics and thus presumably via different pathways (3, 13, 14, 15, 16, 17). Significance of the MC4-R is also supported by association of MC4-R gene mutations with early-onset obesity in humans (18, 19, 20). {alpha}- and {gamma}3-MSH are cleaved from POMC in the hypothalamus. {alpha}-MSH is an agonist for both MC4- and MC3-Rs. It seems to be the mediator of the effects of POMC on body weight, as administration of an {alpha}-MSH analog reverses the obesity syndrome in POMC null mice (4). The function of {gamma}3-MSH in energy balance control is not known, but because it is a strong agonist for the MC3-R, it most likely is of importance in controlling body weight.

Although there is strong and conclusive evidence in both rodents and humans that inactivation of the melanocortin system will lead to obesity, there is little evidence that prolonged activation of the system will decrease weight and could thus be used for the treatment of obesity. Acute administration of {alpha}-MSH reduces food intake in rodents, but long-term treatment with {alpha}-MSH is problematic due to the short half-life of {alpha}-MSH peptide and the requirement for central administration. To overcome this problem, we decided to use a transgenic approach and generated mice overexpressing {alpha}-MSH and {gamma}3-MSH. The transgene encodes N-terminal POMC and is under the control of the cytomegalovirus (CMV) promoter, which drives the expression of these genes in almost all tissues. However, N-terminal POMC requires further processing to produce mature and active peptides, such as cleavage by prohormone convertases 1/3 and 2 (21, 22, 23) and C-terminal amidation by peptidylglycine {alpha}-amidating monooxygenase (24). We hypothesized that biologically active peptides would be derived from the transgene in a limited set of cell types, i.e. cells in the hypothalamus, pituitary, and skin, among others that synthesize and secrete MSH. Because unprocessed N-terminal POMC has little bioactivity, a form of tissue specificity for transgene expression might be achieved based on the necessity for posttranslational processing to produce bioactive MSH.

Mouse models of melanocortin inactivation (Ay, POMC, and MC4-R knockout mice) show increased weight gain, linear growth, food intake, and feeding efficiency. In addition, Ay and MC4-R knockout mice develop insulin resistance and hyperinsulinemia and have signs of defects in glucose metabolism even before the obesity develops (25, 26). Based on these findings we hypothesized that MSH-overexpressing mice would have decreased weight gain, linear growth, adiposity, and food intake and improved insulin sensitivity. We also postulated that these effects would be more pronounced in obesity models (Ay, db3J/db3J) whose obesity is partially attributable to disturbances in melanocortin signaling.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Transgene construction
Rat hypothalamic cDNA was amplified with primers rMSH1 (5'-gtcctcagaaagcttcctttccgc-3') and rMSH2 (5'-cacagggcgaagcttctagcccaccgg-3') that are complementary to the rat POMC with some modifications. The amplified fragment includes part of the 5'untranslated region, the signal sequence, the sorting sequence, {gamma}3-MSH, the joining peptide, and {alpha}-MSH, including the C-terminal glycine necessary for amidation (Fig. 1Go). The codon for the C-terminal glycine is immediately followed by a stop codon. The amino acid sequences for the rat and mouse {alpha}-MSH and {gamma}3-MSH peptides are identical. The DNA fragment was cloned into SmaI site of an expression vector pCI-neo (Promega, Madison, WI), which contains the human CMV immediate-early enhancer/promoter region, a rabbit ß-globin/IgG chimeric intron, and simian virus 40 (SV40) late polyadenylation signal. The transgene construct was excised using BglII and BamHI and purified from the vector with two cycles of gel purification.



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  		FIG. 1. A, Structure of the MSH transgene containing human CMV immediate-early enhancer/promoter region (CMV promoter/enhancer), a rabbit ß-globin/IgG chimeric intron (synthetic intron), 79-bp 5'untranslated region, signal sequence, sorting sequence, {gamma}3-MSH, joining peptide (JP), and {alpha}-MSH fragment of the POMC gene (MSH cDNA) and SV40 late polyadenylation signal (SV40 polyA signal). B, Structure of the POMC gene showing the MSH transgene in gray.

 
Production of transgenic mice
DNA was microinjected into fertilized C57BL/6 x CBA F2 oocytes. Surviving oocytes were transferred to the oviducts of pseudopregnant foster mothers according to previously described methods (27). These procedures were performed by the Herbert Irving Comprehensive Cancer Center Transgenic Mouse Facility at Columbia University. Eighteen progeny were obtained, four of which were positive for the transgene by PCR analysis of ear-clip DNA. The founders were mated with mice from a congenic B6 strain (C57BL/6J.129-AwJ db3J) recently constructed by Chua and colleagues (28, 29). This is a white-bellied agouti-colored line, which was used instead of black mice to be able to visualize the potential darkening effect of MSH on coat color. Four founders transmitted the transgene to their progeny. The expression of the transgene was evaluated in progeny by RT-PCR analysis using primers based on sequence after the CMV transcription start site (CMV-start, 5'-cagtgcttctgacacaacagtctc-3') and a sequence in the end of the MSH-fragment (rMSH-2). Amplification from cDNA produced a 701-bp fragment and from genomic DNA a 833-bp fragment. Two lines (nos. 7 and 10) were shown to express mRNA for the transgene. The transgene expression in line 7 was weak with extremely patchy expression in the skin and low levels of expression in the hypothalamus, and therefore the line was not used for further analysis. One line 10 N1 mouse was mated with C57BL/6J.129-AwJ db3j/+ and N2 mice with B6.Cg-Ay/J. N3 mice were intercrossed to get transgene homozygote and nontransgenic mice that were also Ay hemizygote or db3J homozygote for these mutations (Fig. 2Go). N3 mice, which had been backcrossed for three generations to C57BL/6J.129-AwJ were used for the phenotype analysis of lean vs. Ay and db3J obesities. For the analysis of the lean mice, the mice were backcrossed to C57BL/6J.129-AwJ strain for three more generations and N6 was used for the analysis of the lean phenotype. Transgene mRNA and peptide expression was initially determined in multiple tissues in the hemizygote mice (Fig. 3Go and Table 1Go). Subsequently peptide expression was quantified in various brain regions in both hemi- and homozygote mice (Fig. 4Go). Obesity and diabetes phenotype measures were performed in transgenic homozygote mice.



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  		FIG. 2. Picture of nontransgenic (+/+), MSH transgene hemizygous (Tg/+), and homozygous (Tg/Tg) mice in agouti-colored AwJ background (A) and yellow-colored Ay background (B).

 


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  		FIG. 3. MSH transgene mRNA in line 10 hemizygous mice by RT-PCR showing mRNA expression (701 -p band) in hypothalamus (Hypo), brainstem (BS), cerebellum (Cb), adrenal (Adr), skin, muscle (Mus), liver (Liv), kidney (Kid), lung, heart (Hrt), stomach (Stom), and intestine (Int). There was no expression in cortex (Cx) or ovary (Ova); + and – controls are shown on the right. Transgene mRNA was also highly expressed in the pituitary (data not shown).

 

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  		TABLE 1. Concentration of POMC-derived peptides in MBH, brainstem, cerebellum, heart, skeletal muscle, kidney, and blood in nontransgenic (+/+) and MSH transgene hemizygous (Tg/+) mice (n = 5–6/group)

 


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  		FIG. 4. Ratios of {gamma}3-MSH and {alpha}-MSH to ß-endorphin in the MBH and brainstem of nontransgenic (+/+), transgene hemizygous (Tg/+), and homozygous (Tg/Tg) mice. *, P < 0.05, transgenic compared with nontransgenic, n = 4–10/group.

 
All animals used were maintained under barrier condition at 22 C with a 12-h light, 12-h dark cycle (0700–1900 h). Animals had free access to rodent food (Picolab 5058; PMI Feeds Inc., St. Louis, MO) and water unless specified. Pathogen testing is done on a quarterly basis, and all serologies have been negative to date.

Analysis of genomic DNA
Transgenic mice were identified using PCR analysis of genomic DNA isolated from ear clips or tail clips. PCR was carried out with primers CMV-start and rMSH-2. The Leprdb3J mutation was identified using primers Ex-11R (5'-gttcttcagtcacgcttga-3') and Lepr-53 (5'-catgaggtattcgatgcaaag-3'). Genotype at the agouti locus was based on the presence of a complete (a) or partial retroviral insertion (AwJ) in intron 1 (30). The A, Ay, and AwJ alleles were identified with primers 39AgouinR (5'-aacctgtgtgaaaccctggg-3') and 38AgouEx1F (5'-cggaatagagtcacttgt gct-3') that gave a 1.4-kb fragment for the A and Ay alleles, 2 kb for AwJ, and no product for a. Primers 43AgoIntSen (5'-ctctcttcggttctgacttga-3') located in the agouti intron 1 and 42LTRrev (5'-cgtcttgagggatcactaca-3') located in the LTR present in a allele were used to genotype for a (700 bp). Because A and Ay alleles give the same size products, mice carrying agouti allele A were identified by visual inspection of the belly hair color because transgenic Ay mice have a distinctly lighter shade of hair, compared with transgenic A mice. Only transgenic hemizygote yellow mice with genotype Ay/AwJ or Ay/a and transgene hemizygote normal mice with no A were used for further matings.

Hair total melanin content
Total melanin content of hair was measured using a spectrophotometric assay described earlier (31). Sepia melanin (Sigma, St. Louis, MO) was used as a standard for the assay.

Measurement of {gamma}3-MSH, {alpha}-MSH, and ß-endorphin
{gamma}3-MSH, {alpha}-MSH, and ß-endorphin were assayed using RIA as described earlier. {gamma}3-MSH RIA was performed with an antiserum provided by Drs. H. Akil and J. Meador-Woodruff (University of Michigan School of Medicine, Ann Arbor, MI) directed against a midportion region of {gamma}3-MSH (32). This assay detects two forms of {gamma}3-MSH immunoactivity in the rat hypothalamus, a large molecular weight form and fully processed, glycosylated {gamma}3-MSH (33). {alpha}-MSH was measured with an antiserum raised in this laboratory that cross-reacts fully with desacetyl {alpha}-MSH but does not cross-react with ACTH or corticotropin-like intermediate-lobe peptide (34). The antiserum does not cross-react with the free acid form of {alpha}-MSH, which has not been amidated. Thus, the {gamma}3-MSH assay detected {gamma}3-MSH immunoactivity in most regions that expressed the transgene, whereas the {alpha}-MSH assay detected only {alpha}-MSH in regions capable of proper processing and amidation. ß- Endorphin was assayed with an antiserum raised in this laboratory directed at ß-endorphin 18–25 (34). This antiserum does not cross-react with {alpha}-MSH, ACTH, or {gamma}3-MSH and would not be expected to detect any peptide products produced by the transgene. Peptide levels were measured from tissue samples homogenized in 0.1 N HCl and then centrifuged at 4000 x g. The supernatant was diluted with assay buffer and used for RIA. Protein content was determined by the Bradford method using BSA as the standard. In addition, the supernatant pooled from medial basal hypothalamus (MBH), brainstem, and cerebellum homogenates from three nontransgenic and three transgenic mice were chromatographed on G-75 Sephadex columns as described earlier, and the eluted fractions were assayed for {alpha}-MSH and {gamma}3-MSH immunoactivity (33).

Isolation of mRNA and quantification by solution hybridization assay
The MBH was dissected as described previously in the rat (35) using a mouse brain matrix. A 2-mm coronal section caudal to the optic chiasm was used. The total RNA was extracted by the Rneasy minikit (Qiagen, Valencia, CA) according to the manufacturer’s instructions and quantified by spectrophotometry. RNA used to generate standard curves and 32P-labeled RNA probes were synthesized using commercial transcription kits (Promega). Transcription was performed from plasmid vectors containing T3 and T7 promoters and either a 254-bp mouse AGRP amplified from mouse brain cDNA using primers Agrp1 (5'-agggcatcagaaggcctgaccagg-3') and Agrp2 (5'-acgtgctactgccgcttcttcaag-3') and cloned into SmaI site of Bluescript vector (pBS+) or a 923-bp mouse POMC cDNA fragment (pMKSU16) (36). POMC probe targeted the sequence after the sequence included in the transgene so that only changes in the endogenous POMC expression were detected. Therefore, the plasmid pMKSU16 was linearized with NcoI at amino acid 377 for the transcription of the RNA probe. Sense RNAs were quantified spectrophotometrically and used to generate standard curves in the hybridization assays. Components of the hybridization reaction included cytoplasmic RNA in 5 µl H2O; 24.5 µl hybridization buffer containing 80% formamide, 40 mM PIPES (pH 7.4), 400 mM NaCl, 1 mM EDTA; 5 µg yeast RNA; and 32P-labeled riboprobe (37). Reaction mixtures were heated at 85 C for 5 min and then incubated for 16 h at 45 C. After hybridization, 300 µl S1 buffer [300 mM NaCl, 30 mM NaAc (pH 4.8), 3 mM ZnCl2, 20 µg/ml salmon sperm DNA] and 1200 U S1 nuclease (Roche Molecular Biochemicals, Mannheim, Germany) were added and incubated for 1 h at 56 C. Reaction mixtures were phenol-chloroform extracted, precipitated, resuspended in TE, and heated for 5 min at 68 C before electrophoresis on a 4% nondenaturing acrylamide gel. Protected bands were quantified by visualization with an autoradiogram followed by excision of the corresponding bands from the gel and liquid scintillation counting (1500 analyzer, Packard, Meriden, CT) and comparison with the standard curve. Because the protected hybrids were smaller than the cellular transcripts, 32P counts were normalized to the full-length RNA species: 0.7 kb for full-length mouse AGRP cytoplasmic RNA (5); 1.0 kb for full-length mouse POMC cytoplasmic RNA (36). Results are presented as picograms cytoplasmic RNA per microgram total RNA.

Measurement of blood glucose and insulin
Tail blood for glucose and insulin was collected from fed and/or 24-h fasted animals in the morning. The mice were lightly restrained, and only the first 5 µl were used for glucose measurements. Glucose was measured using the glucose-oxidase method (Glucometer Elite; Bayer, Elkhart, IN). Serum insulin was quantified using a commercial RIA kit from Linco (St. Louis, MO).

Glucose tolerance test (GTT)
After a 24-h fast, glucose (1 mg/g) was administered ip and blood glucose measured from tail blood before and 20, 40, 60, and 90 min after glucose administration. A second test was performed later to collect blood (30 µl) for insulin measurements at 0 and 20 min time points.

Cold tolerance test
Core (rectal) temperature of individually housed mice was measured using a thermocouple probe (IT-18, Physitemp) connected to a thermocouple thermometer (BAT-10; Physitemp, Clifton, NJ) at 22 C ambient and 20 and 40 min after being placed at 4 C ambient.

Measure of food intake
Mice were separated 4 d before measurements into two to three mice of the same genotype/cage. There were three to four cages per group. Weighed pellets of food were placed in the cage, and remaining food was weighed 72 h later. Values are means from two measurements and are expressed as grams per 24 h per mouse. Food intake after a 24-h food deprivation was measured in the morning 24 h after the start of refeeding.

Weight and nasoanal length (NAL)
Mice were weighed weekly. NAL was measured at 3 months of age (Ay and db3J) and at 6 months (lean), 5–6 months (db3J), 6–7 months (Ay female), and 7–8 months Ay (male).

Body fat analysis
A dual-energy x-ray absorptiometry (DEXA) scan was used to obtain the total body fat percentages (Lunar PIXImus, Lunar, Madison, WI).

Statistical analysis and calculations
Quantitative insulin sensitivity check index (QUICKI) was calculated from fasting insulin (milliunits per milliliter) and glucose (milligrams per deciliter) values as 1/[log(insulin) + log(glucose)] as described by Katz et al. (38). Statistical analysis was carried out with Student’s t test when only two groups were compared for instance transgenic and nontransgenic mice of the same obesity genotype and sex. Two-way ANOVA was used to determine the effects of transgene and sex or obesity genotype. One-way ANOVA followed by Fisher’s protected least difference or Tukey tests were used to evaluate the significance of the differences between individual groups in such cases. ANOVA for repeated measures was used when values for different time points were available. P < 0.05 was considered statistically significant. The results are reported as mean values ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Transgene expression
The mRNA expression of the transgene was measured with RT-PCR using transgene specific primers. Line 10 hemizygous mice showed transgene expression in most of the tissues studied (Fig. 3Go). The brain regions expressing the transgene mRNA were the hypothalamus, brainstem, and cerebellum, whereas no expression was detected in the cortex. Peripheral tissues including adrenal, skin, muscle, liver, kidney, heart, stomach, and intestines, but not the ovaries, expressed the transgene mRNA. In a separate study, transgene mRNA was also found to be strongly expressed in the pituitary of line 10 homozygous mice.

MSH peptide levels were measured by RIA using antibodies that detected both endogenous and transgene-derived {alpha}- and {gamma}3-MSH. Because the transgene did not encode ß-endorphin, the level of ß-endorphin was also determined and used as a marker for endogenous POMC peptide production. MSH overexpression did not change ß-endorphin levels in any tissues measured. The ratios of {gamma}3-MSH to ß-endorphin and {alpha}-MSH to ß-endorphin were significantly increased in the MBH and brainstem of the transgenic compared with nontransgenic mice (Fig. 4Go). {alpha}-MSH and {gamma}3-MSH were increased 1.5- to 2-fold in the MBH and 2- to 4-fold in the brainstem of transgene homozygous mice. The peptide levels in other tissues are shown in Table 1Go. There were significantly increased levels of both {gamma}3-MSH and {alpha}-MSH in the heart and blood, but only {gamma}3-MSH was increased in the skeletal muscle and kidney. Because the {alpha}-MSH antibody is specific for the properly processed, amidated peptide, transgene-derived {alpha}-MSH expression is detected only in tissues in which {alpha}-MSH can be normally processed (MBH, brainstem, heart). In contrast, tissues that cannot process POMC completely (cerebellum, skeletal muscle, kidney) showed only increased {gamma}3-MSH. Samples from the MBH and cerebellum of transgenic and nontransgenic mice were chromatographed on G-75 Sephadex columns, and the fractions were assayed for {alpha}-MSH and {gamma}3-MSH by RIA (Fig. 5Go). The elution patterns for both {alpha}-MSH and {gamma}3-MSH were identical in MBH samples from transgenic and nontransgenic mice. All of the {alpha}-MSH immunoreactivity eluted in the same position as the synthetic {alpha}-MSH standard. As reported previously in the rat hypothalamus (33), the {gamma}-MSH immunoactivity in the MBH eluted in two peaks with the second peak corresponding to the elution of fully processed glycosylated {gamma}3-MSH and the first peak corresponding to larger molecular weight material, which eluted just after the ß-lipotropin standard. In contrast, in the cerebellum, no {alpha}-MSH immunoactivity was detected, and all of the {gamma}3-MSH immunoactivity eluted as a single large molecular weight peak, consistent with unprocessed prohormone, with no immunoactivity eluting in the position of {gamma}3-MSH. Thus, although the transgene was expressed in the cerebellum, neither {alpha}-MSH nor {gamma}3-MSH was produced.



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  		FIG. 5. Sephadex G-75 chromatography of the {alpha}-MSH immunoactivity in the MBH (upper panel) and {gamma}3-MSH immunoactivity (lower panel) in the MBH and cerebellum of line 10 transgenic (Tg) mice. All of the {alpha}-MSH immunoactivity in the MBH eluted in the same position as the synthetic {alpha}-MSH standard. There was no detectable immunoactivity with this antiserum in the cerebellum. The {gamma}3-MSH immunoactivity in the MBH of the transgenic mice was processed normally and eluted in two peaks, with the second peak corresponding to the elution position of glycosylated {gamma}3-MSH. In the cerebellum, however, only a single large molecular peak was detected with no immunoactivity eluting in the position of {gamma}3-MSH.

 
Transgene effects on melanin production
Transgenic MSH overexpression led to a uniformly dark coat color in agouti-colored C57BL/6J.129-AwJ mice (Fig. 2Go). There was a gene-dosage effect such that the transgenic homozygotes were clearly darker than the hemizygotes. MSH overexpression also led to dark coat color in B6.Cg-Ay/J background. In fact, transgenic homozygous Ay mice were indistinguishable in color from their transgenic homozygous AwJ/a littermates. Therefore, we had to develop a new genotyping protocol for Ay as described in Materials and Methods. We measured the total melanin content of the hair using a spectrophotometric assay. Total melanin content of agouti-colored nontransgenic and homozygous transgenic hair was not different (Table 2Go). The yellow hair of Ay mice had one fifth of the total melanin content of agouti hair, and hair of transgenic Ay mice had a similar melanin content as the hair of agouti mice. The MSH effects on coat color and melanin content confirm the biological activity of the MSH peptide produced by the transgene. Furthermore, MSH was produced in a sufficient amount to counteract agouti overexpression in the skin of yellow Ay mice.


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  		TABLE 2. Concentration of total melanin in dorsal hair of nontransgenic (+/+) and homozygote MSH overexpressing (Tg/Tg) agouti-colored mice and yellow Ay mice

 
Transgene effects on body weight and metabolism
Body weight and composition, food intake, glucose and insulin levels, and glucose tolerance were studied in lean transgenic homozygous and nontransgenic N6 mice. Similar measurements were also made in N3 lean vs. Ay and db3J/db3J mice with and without homozygous transgene expression. In addition, a cold tolerance test was performed in N3 db3J/db3J mice with and without the transgene. POMC and AGRP mRNA was measured in the hypothalamus of the N3 lean and Ay mice with and without the transgene.

Body weight, NAL, and adiposity
Lean mice.
MSH overexpression significantly decreased weight gain in lean male mice (P = 0.011, ANOVA for repeated measures, n = 12 Tg/Tg; n = 18 +/+) but had no effect on weight in the female mice (P = 0.72, n = 20 Tg/Tg; n = 14 +/+) (Fig. 6Go). The weight difference in the males was statistically significant from the age of 7 wk (P = 0.04) and was on average 4.8 g or 13% in 26-wk-old mice (P = 0.004). The MSH-overexpressing male mice had shorter NAL (P = 0.02, Table 3Go) with decreased fat percentage (28.6 ± 1.8 vs. 33.5 ± 1.5%) measured with DEXA (P = 0.04, Table 3Go). There were no differences in NAL or fat percentage in the females.



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  		FIG. 6. Growth curves of nontransgenic and transgenic db3J/db3J, Ay, and lean male and female mice (n = 9–20/group).

 

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  		TABLE 3. Nasoanal length and whole-body fat percentage measured with DEXA in nontransgenic (+/+) and homozygote MSH overexpressing (Tg/Tg) lean, Ay, and db3J/db3J female and male mice at the age of 6 months

 
db3J/db3J Mice.
The MSH transgene significantly decreased weight gain in db3J/db3J male and female mice (Fig. 6Go). The weight difference was apparent at 5 wk of age in females (P = 0.026) and 13 wk in males (P = 0.023). There were no differences in NAL in 3-month-old female or male transgenic compared with the nontransgenic db3J/db3J mice. A subset of the mice (n = 6–11/group) was killed at the age of 5–6 months. The female transgenic mice weighed 13 g and the male mice 5 g less than the controls. The female mice were significantly shorter than the controls (P = 0.04, Table 3Go). The whole body fat percentage obtained with DEXA was decreased significantly in the males (P < 0.001) but not in the females (P = 0.10, Table 3Go).

Ay Mice.
Transgenic Ay female mice weighed less than their nontransgenic Ay controls from the seventh week onward (P = 0.03, t test at 7 wk, P = 0.042, ANOVA for repeated measures age 6–25 wk). At 3 months transgenic Ay females were on average 0.5 cm shorter (P = 0.0001) and weighed 5 g less than controls (P = 0.009). There was no difference in weight gain between transgenic and nontransgenic Ay males (P = 0.29). At 6–7 months (females) or 7–8 months (males), the mice were killed. MSH-overexpressing female Ay mice still weighed 5 g less (P = 0.026) and were shorter (P = 0.023) than the controls but had no difference in fat percentage measured with DEXA (Table 2Go). There were no differences in weight, NAL, or fat percentage between MSH-overexpressing male Ay mice and the controls.

Food intake
No significant difference in basal 24-h food intake was detected between MSH transgenic and nontransgenic controls in lean (N3 or N6) or obese mice. The lean female mice ate 3.2 ± 0.2 g (+/+) and 3.2 ± 0.3 g (Tg/Tg) and males 3.5 ± 0.1 g (+/+) and 3.9 ± 0.3 g (Tg/Tg). Female Ay mice ingested significantly more than their N3 lean controls (4.2 ± 0.4 g, P = 0.02 Ay +/+, compared with lean +/+ female), but there was no difference between +/+ and Tg/Tg Ay females (4.0 ± 0.1 g, Tg/Tg Ay female). Similarly, food intake was increased in Ay male mice (4.8 ± 0.2 g), compared with N3 lean controls (P = 0.001 Ay +/+, compared with lean +/+ male), but was not changed by the MSH overexpression in Ay males (4.4 ± 0.5 g). Food intake was 4.0 ± 0.3 g in nontransgenic and 4.0 ± 0.2 in transgenic db3J/db3J mice.

Food intake for 24 h refeeding after a 24-h fast was also measured. There were no differences between transgenic and nontransgenic mice or between lean and Ay mice. The food intakes were: females, 6.2 ± 0.3 g (+/+ lean), 5.7 ± 0.4 (Tg/Tg lean), 5.6 ± 0.5 g (+/+ Ay), 5.3 ± 0.4 g (Tg/Tg Ay); and males, 5.4 ± 0.2 g (+/+ lean), 5.2 ± 0.2 g (Tg/Tg lean), 5.8 ± 0.1 g (+/+ Ay), 6.0 ± 0.2 g (Tg/Tg Ay). Nontransgenic and transgenic db3J/db3J mice both ingested 4.6 ± 0.2 g.

Cold tolerance
Cold tolerance was tested in db3J/db3J mice. Results from male and female mice were combined. There was no difference between nontransgenic (n = 9) and transgenic (n = 12) mice in the response to cold challenge at 4 C (0 min: 35.3 ± 0.2 C vs. 34.3 ± 0.2 C; 20 min: 32.3 ± 0.7 vs. 32.0 ± 0.5 C; 40 min: 29.9 ± 1.2 vs. 29.0 ± 0.8 C, P = 0.37).

Glucose metabolism
Lean mice.
Fed and fasted plasma insulin was decreased in the female and male MSH-overexpressing mice (P = 0.0009 for females and P = 0.0079 for males, two-way ANOVA; Fig. 7Go), but there was no difference in plasma glucose. There was a significant effect of the transgene on glucose tolerance in the males (P = 0.04, Fig. 8Go) but not in the females. QUICKI was calculated from fasting insulin and glucose values. Transgenic mice had significantly higher QUICKI than nontransgenic controls, consistent with improved insulin sensitivity (females: 0.317 ± 0.002 vs. 0.340 ± 0.005; males: 0.303 ± 0.009 vs. 0.322 ± 0.010, P = 0.007 for genotype, P = 0.037 for sex, and P = 0.71 for genotype x sex interaction, two-way ANOVA).



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  		FIG. 7. Blood glucose (A) and serum insulin (B) values in lean nontransgenic and transgenic males (n = 9/group) and females (n = 9 and 15, respectively) in overnight fasted and fed state. Blood glucose (C) and serum insulin (D) values in Ay nontransgenic and transgenic males (n = 9 and 12, respectively) and females (n = 9 and 9) in baseline fasting state and 20 min after an ip glucose challenge (1 mg/g). *, P < 0.05 transgenic, compared with nontransgenic.

 


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  		FIG. 8. GTT (ip 1 mg/g) in nontransgenic and transgenic lean and Ay male mice. Glucose tolerance was significantly improved in transgenic lean (P = 0.01, ANOVA for repeated measures) and Ay mice (P < 0.001) (n = 17 for non-Tg lean, 8 for Tg lean, and 12/group for Ay.

 
db3J/db3J Mice.
There was no effect of the transgene seen on any measure of glucose metabolism (fed and fasting glucose and insulin and GTT) in male or female db3J/db3J, despite the decrease in body weights due to the MSH overexpression.

Ay Mice.
Compared with the lean controls Ay mice had significantly higher fasting glucose and insulin values. Fasting glucose, measured on two different occasions, was significantly decreased in Tg/Tg Ay males, compared with +/+ Ay males (P < 0.005), but not changed in females (Figs. 7Go and 8Go). In contrast, fasting insulin was not changed in males but was significantly decreased in females (P = 0.04, Fig. 7Go). Fed glucose was not changed in males but was lower in females (159 ± 13.6 +/+ Ay vs. 131 ± 5.4 mg/dl Tg/Tg Ay, P = 0.018). There were no differences in fed insulin levels. Glucose tolerance during GTT was improved in Tg/Tg Ay males (P < 0.01, ANOVA for repeated measures, Fig. 8Go), but there was no difference in insulin release during the GTT in males (Fig. 7Go). In contrast, in the Tg/Tg Ay females, plasma glucose values were not changed during GTT, but stimulated insulin at 20 min was significantly lower (P = 0.03). Transgenic Ay females had significantly higher QUICKI than nontransgenic Ay females, consistent with improved insulin sensitivity (0.291 ± 0.007 vs. 0.272 ± 0.006, P = 0.025). A similar trend was seen with QUICKI in transgenic Ay males (0.282 ± 0.005 vs. 0.272 ± 0.004, P = 0.08).

Hypothalamic POMC and AGRP mRNA expression
There were no effects of transgene or Ay genotype seen on endogenous POMC mRNA levels in the females [+/+ lean (n = 8) 0.30 ± 0.06 pg/µg total RNA, Tg/Tg lean (n = 11): 0.28 ± 0.03 pg/µg, +/+ Ay (n = 10): 0.23 ± 0.02 pg/µg, Tg/Tg Ay (n = 9): 0.26 ± 0.04 pg/µg]. AGRP mRNA was significantly decreased in the Ay, compared with lean (P = 0.04), but there was no effect of the transgene (+/+ lean: 0.26 ± 0.07 pg/µg RNA; Tg/Tg lean: 0.25 ± 0.03 pg/µg; +/+ Ay: 0.18 ± 0.02 pg/µg; Tg/Tg Ay : 0.17 ± 0.04 pg/µg).

There was a significant decreasing transgene effect on POMC mRNA in the male mice: +/+ lean (n = 11), 0.75 ± 0.05 pg/µg RNA; Tg/Tg lean (n = 8), 0.63 ± 0.11 pg/µg; +/+ Ay (n = 7), 0.85 ± 0.09 pg/µg; Tg/Tg Ay (n = 10), 0.53 ± 0.04 pg/µg (P = 0.005, two-way ANOVA). There were no significant changes in AGRP mRNA in the males (+/+ lean: 0.57 ± 0.07 pg/µg RNA; Tg/Tg lean: 0.62 ± 0.14 pg/µg; +/+ Ay: 0.67 ± 0.13 pg/µg; Tg/Tg Ay : 0.49 ± 0.09 pg/µg).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The aims of this study were to produce a transgenic mouse overexpressing {gamma}3- and {alpha}-MSH and characterize the effects of overexpression on the obesity and diabetes phenotypes in lean and genetically obese mice. One mouse line showed appropriate transgene expression and was chosen for the phenotype characterization. In this line, the transgene mRNA is expressed ubiquitously as expected when using the CMV promoter. It is translated in many tissues including the MBH as evidenced by increased {gamma}3- and {alpha}-MSH peptide levels. Furthermore, the RIA and chromatography results demonstrated that the peptides are processed properly in the tissues that normally process MSH peptides. The change in coat color confirms that the MSH produced from the transgene is biologically active. The level of overexpression is sufficient even to overcome the effects of overproduction of agouti on coat color and melanin content in Ay yellow mice. We believe that the effects on coat color and melanin production are due to local production of MSH in the skin rather than an effect of elevated MSH in the circulation. This is due to our observation in a second transgenic line (no. 7) that showed dark patches of fur in transgenic Ay mice (data not shown). If the increased eumelanin production was due to elevated circulating MSH, then one would have expected a uniform darkening of coat color rather than the observed patchy distribution.

The effect of MSH overexpression on body weight and body composition was studied in MSH transgene homozygote lean and obese db3J/db3J and Ay mice. MSH overexpression in lean mice resulted in decreased weight gain and adiposity in male mice but had no effect in female mice. MSH overexpression decreased weight gain and adiposity in obese, leptin receptor-deficient, female and male db3J/db3J mice. In contrast, adiposity was not reduced in Ay mice. The female Ay mice, however, weighed less than their nontransgenic controls and the increased NAL characteristic of the Ay mouse was attenuated by the transgene.

The predicted mechanisms by which MSH overexpression decreases body weight would be a decrease in food intake and/or an increase in energy expenditure. We were not able to detect any changes in food intake either in the basal state or on refeeding after a 24-h fast. However, we used a fairly crude method, and it might not have shown small changes that could over a long period of time lead to a significant change in weight gain. Alternatively, the reduction in feeding might have taken place very early on, before we started measuring food intake. Interestingly, hyperphagia is more evident in MC4-R knockout mice when placed on a high-fat diet (17). It remains to be seen in future studies whether MSH overexpression can attenuate the hyperphagia and obesity associated with the ingestion of a high-fat diet.

It is possible that changes in energy expenditure are responsible for the decrease in body weight and adiposity seen in the male MSH transgenic lean mice. Analysis of MC4-R knockout mice indicates that this receptor is involved in stimulating diet-induced thermogenesis and physical activity (3). There is evidence that the central melanocortin system regulates sympathetic nervous system activity to brown adipose tissue (39, 40). The db3J/db3J mice have a defect in sympathetic nervous system and thus impaired brown adipose tissue function leading to poor cold tolerance. Improved cold tolerance in MSH-overexpressing mice would have suggested increased sympathetic activity and energy expenditure, which could have contributed to decreased weight. However, cold tolerance was not improved in the transgenic db3J/db3J mice.

MSH overexpression improved insulin sensitivity in lean and Ay mice. Both the lean and Ay male mice showed improved glucose tolerance. Fed and fasted plasma insulin levels were decreased in lean mice of both sexes and female Ay mice. It is noteworthy that these changes occurred without changes in adiposity in the obese Ay mice and lean females. The lean male mice had decreased adiposity that could contribute to improved glucose tolerance and decreased insulin levels. In contrast, MSH overexpression did not improve hyperglycemia, hyperinsulinemia, or glucose tolerance in db3J/db3J mice, although it did reduce obesity. Others have shown that the melanocortin system may have effects on glucose metabolism independent of effects on feeding and body weight (26, 41). Fan et al. (26) showed that a centrally administered melanocortin agonist inhibited plasma insulin release. Obici et al. (41) showed that a week-long central infusion of {alpha}-MSH enhanced the actions of insulin on glucose uptake and production during insulin clamp studies in the rat, whereas the MC-R antagonist, SHU9119, had an opposite effect. This occurred even though food intake and body weights were similar in {alpha}-MSH and pair-fed SHU9119 groups, although {alpha}-MSH decreased visceral fat mass. These results are consistent with our findings of improved glucose tolerance in both lean and Ay male mice that express the MSH transgene. Although no changes in ip glucose tolerance were noted in the female transgenic mice, plasma insulin levels were significantly lower in these mice. Using QUICKI as an index of insulin sensitivity, both transgenic male and female mice had significantly higher values, consistent with improved insulin sensitivity.

Even though there was an effect of MSH overexpression on obesity, a larger effect could have been expected. One explanation could be that the increase in MSH content in the appropriate areas was not high enough. The CMV promoter is expected to cause ubiquitous expression of the transgene. This seemed to be the case in the line studied. We detected mRNA expression and increased MSH content in many different tissues. At least in the skin in which the expression was clearly visualized, it was very even. There was high enough expression in the skin to cause an almost complete black coat color, even in the yellow Ay mice. The MBH, which includes the most crucial nuclei for the MSH effects on energy metabolism, had 2-fold increased levels of {alpha}-MSH. However, it could be argued that {alpha}-MSH shows a higher affinity for human MC1-R than for MC3-R and MC4-R, whereas AGRP has a higher affinity for MC4-R than {alpha}-MSH (42). These differences could explain a stronger effect in the skin than in the brain with equivalent {alpha}-MSH overexpression. Forbes et al. (43) showed that peripheral injections of an MSH analog reduced weight gain and improved cold tolerance in ob/ob mice, possibly through direct effects on adipose tissue. If these peripheral mechanisms are indeed important, they should have been activated in the MSH transgenic mice because there was an almost 2-fold increase in circulating MSH peptides, even in the transgene hemizygote mice. Another explanation for the modest effects on body weight could be that the body is compensating for the increased MSH. Indeed, endogenous POMC mRNA was decreased in the transgenic Ay males, which might have contributed to unchanged weight gain. In contrast, the female transgenic Ay mice that showed reduced body weight did not have any differences in POMC mRNA levels. We also measured AGRP mRNA. In line with an earlier report (5), AGRP mRNA was reduced in the female Ay mice, compared with the lean controls. There was no change in AGRP mRNA levels in the transgenic mice, but this does not rule out other compensatory changes in AGRP peptide release or activity. Furthermore, other compensatory mechanisms such as changes in the melanocortin receptors or other neuropeptide systems cannot be ruled out.

In general it has been easier to demonstrate stimulatory effects of AGRP and other MSH antagonists on feeding than to demonstrate inhibitory effects of MSH agonists. There is evidence that AGRP is an inverse agonist for MC4-R and that MC4-R has some constitutive activity (44, 45). These findings give rise to the hypothesis that constitutively active MC4-R tonically suppresses feeding, which is reversed by AGRP even in the absence of the agonist MSH. This hypothesis raises questions about the ability of increased MSH to inhibit feeding and reduce weight in the basal state because the receptor is already activated. Our results show that increased MSH can reduce weight both in lean and obese mice. Thus, MC4-R may have some constitutive activity, but increased MSH is able to further activate it.

Recently another POMC transgenic mouse model was published by Mizuno et al. (46). In this model the whole POMC gene was expressed specifically in neuronal cells under the control of the neuron-specific enolase promoter. Consistent with our results, this transgene attenuated obesity in ob/ob mice, but unlike our results in db3J/db3J mice, the diabetes phenotype of the ob/ob mice was almost completely reversed by the POMC transgene. Similar to our results, there was a small effect on body weight in transgenic lean mice, but in contrast to our study results, POMC overexpression did not affect insulin levels or improve glucose metabolism in lean mice. Furthermore, the transgene decreased fasting-induced hyperphagia in lean mice. However, the authors reported 1-h refeeding, whereas we measured 24-h refeeding. The differences in results from POMC overexpressing and our {alpha}- and {gamma}3-MSH-overexpressing mice could be attributed to the inclusion of ß-endorphin, ß-MSH, and ACTH in the POMC transgene. ß-Endorphin has complex effects on feeding and can stimulate feeding when injected intracerebroventricularly, but in contrast, mice selectively lacking ß-endorphin become obese (47). It is thus possible that ß- endorphin overexpression contributed significantly to the phenotype of the POMC transgenic mice. A significant role for ß-MSH in inhibition of feeding via MC4-R has also been proposed (48). Thus, there were a number of experimental differences between our study on {alpha}- and {gamma}3-MSH-overexpressing mice and the study by Mizuno et al. (46) on POMC overexpressing mice, including promoter, genetic background, age, and time of feeding studies. It remains to be determined whether the lack of effect on hyperphagia in db3J/db3J mice or fasting-induced hyperphagia in lean mice is due to these differences or whether ß-endorphin and/or ß-MSH play a crucial role in decreasing food intake in POMC-overexpressing mice. However, the obesity and diabetes attenuating effects of the whole POMC gene and {alpha}- and {gamma}3-MSH alone are quite similar, and it therefore appears that these peptides are responsible for most POMC effects on regulation of body weight and glucose metabolism.

Although significant effects of transgene overexpression were noted in our study in both male and female mice, a number of sex differences emerged. The most notable difference was seen in body weight and body composition in the lean transgenic mice, in which the females resisted weight loss. Interestingly, in the study by Mizuno et al. (46), the effect of the POMC transgene was more pronounced in the lean males, compared with the lean females. Sex differences were also noted in another study of heterozygote POMC null mice, in which male mice, but not female mice, developed late-onset obesity (49). All of these studies including our own were performed in C57BL/6J mice, which have well-known sex differences with respect to metabolic phenotypes, especially in response to diet-induced obesity. In addition, sex steroids have well-documented effects on POMC expression in the hypothalamus (50, 51). It remains to be determined how sex steroids and POMC may interact with respect to obesity and metabolic phenotypes.

In summary, our results show that MSH overexpression was effective in reducing weight gain and adiposity and improving glucose tolerance and insulin sensitivity in lean mice and in genetic mouse obesity models. Additional study is necessary to determine whether MSH overexpression will protect against more common forms of obesity such as diet-induced obesity and the associated metabolic changes. The results of the current study, however, provide further support for the hypothesis that long-term melanocortinergic activation could serve as a potential strategy for antiobesity and/or antidiabetic therapy.


   Acknowledgments
 
The technical assistance of Irene Conwell is greatly appreciated.


   Footnotes
 
This work was supported by National Institutes of Health Grants DK57561 (to S.L.W.), DK57621, and DK26687 (to S.C.C.) and Jalmari and Rauha Ahokas Foundation and Academy of Finland grants (to E.S.).

Abbreviations: AGRP, Agouti-related peptide; CMV, cytomegalovirus; DEXA, dual-energy x-ray absorptiometry; GTT, glucose tolerance test; MBH, medial basal hypothalamus; MC-R, melanocortin receptor; NAL, nasoanal length; POMC, proopiomelanocortin; QUICKI, quantitative insulin sensitivity check index; SV40, simian virus 40.

Received March 2, 2004.

Accepted for publication April 19, 2004.


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