Heterozygous leptin deficient mice

All animal studies were performed according to procedures approved by The University of Massachusetts Medical School’s Institutional Animal Care and Use Committee. Hlx heterozygous knockout mice (Stock# 008315) and heterozygous leptin-deficient mice (Stock# 000632) were purchased from Jackson Laboratory. Full-length Hlx cDNA was fused downstream with the 5.4-kb aP2 (Fabp4) promoter. The transgenic DNA fragment was gel-purified and injected into fertilized embryos harvested from C57BL/6 × SJL hybrid mice. Transgenic lines were backcrossed with C57BL/6 for at least three generations. Normal diet containing 4% (w/w) fat and high-fat diet containing 36% (w/w) fat were purchased from Harlan Teklad and Bioserv (Product# F3282), respectively. Food consumption was measured daily for individually caged mice. β3-adrenergic agonist CL-316243 (Tocris, Cat#1499) was intraperitoneally injected into mice at 0.5 mg kg⁻¹ body weight. For glucose tolerance test, glucose was intraperitoneally injected into mice at 2 g kg⁻¹ body weight after 16-hr fasting. For acute cold exposure, mice were individually caged and placed in a 4 °C cold room, and core body temperature was measured with the Microtherma 2 rectal probe (Thermoworks). Gender-matched littermate controls were used in all the experiments of Hlx knockout and transgenic mouse studies.