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Volumn 278, Issue 5343, 1997, Pages 1641-1644

Independent and additive effects of central POMC and leptin pathways on murine obesity

Author keywords

[No Author keywords available]

Indexed keywords

LEPTIN; PROOPIOMELANOCORTIN;

EID: 0030678119     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.278.5343.1641     Document Type: Article
Times cited : (214)

References (32)
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    • note
    • ob/+ or +/+). PCR primers were 5′-ATGAATTCAGGAAAATGTGCTGGAGACCCCTGT and 3′-CAGTCGGTATCCGCCAAGCAG. PCR products were denatured at room temperature for 30 min in 0.2 N NaOH, 15 mM tris-HCI (pH 7.5), and 3.75 mM EDTA, then blotted onto nylon membranes (Bio-Rad, Hercules, CA). Membranes were prehybridized at 50°C for 30 min in 5× standard saline citrate (SSC) and 0.1% SDS, then hybridized with labeled oligonucleotide probes specific for the lepob mutation or wild-type sequence for 1 hour. Membranes were washed in 2× SSC and 0.1% SDS at room temperature for 10 min followed by 3 M tetramethyl ammonium chloride, 50 mM tris-HCI (pH 8), and 0.2% SDS at 63°C for 15 min, then exposed to a Phosphorlmager screen overnight.
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    • note
    • Female mice were adrenalectomized at 6 weeks of age with bilateral flank incisions under anesthesia. Mice were given stress doses of dexamethasone in decreasing doses twice daily for 2 days after adrenalectomy and were then maintained on normal saline drinking water supplemented with corticosterone (1 mg/ml). Complete adrenalectomy was confirmed by monthly corticosterone radioimmunoassay (ImmuChem Double Antibody Corticosterone 125-1 RIA Kit; ICN Biomedicals, Costa Mesa, CA) after exposure of the animals to stress (handling and tail clip). Mice were maintained on normal Purina Rodent Chow, given ad libitum, and weighed weekly beginning at 6 weeks.
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    • note
    • Blood was obtained by tail clip each month, starting at 3 months. Mice were fasted 4 hours before an afternoon blood draw. Glucose concentrations were measured by glucose meter (One Touch Profile; Lifescan, Milpitas, CA), and serum corticosterone and insulin concentrations were measured by radioimmunoassay (Rat Insulin RIA Kit; Unco Research, St. Charles, MO).
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    • note
    • Mice were individually housed. Daily food intake and mouse weights were recorded. Mice were given twice daily subcutaneous injections of saline for 3 to 4 days. Saline was then replaced with recombinant human leptin (2.0 mg/kg). Insulin and corticosterone concentrations were measured on the first and last days of the experiment. Two sets of mice were used: 8-month-old adrenalectomized females and 3-month-old nonadrenalectomized males and females. His-tagged recombinant human leptin was expressed in Escherichia coli and purified from inclusion bodies by nickel affinity chromatography. Large-scale refolding was obtained with an Amicon Spiral-wound cartridge. The histidine tag was removed by thrombin cleavage and the leptin was further purified by ion-exchange chromatography. Two independent batches were used for the experiments shown in Figs. 2 and 3. Both batches were greater than 99% pure as assayed by SDS-polyacrylamide gel electrophoresis and contained 0.357 U per milligram of leptin and 1.21 U per milligram of leptin of endotoxin, respectively.
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    • Corticosterone concentrations in nonadrenalectomized mice were obtained within 1 min of handling mice that had been individually housed in covered cages to reduce the effects of stress on corticosterone level.
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    • note
    • We thank L. Van der Ploeg and M. Tota for recombinant human leptin and helpful discussions. Supported by NIH grants to R.D.C. (DK/AR517330) and B.A.B. (DK02404 and HD33703).


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