Objective: To directly ascertain the physiological roles in adipocytes of hormone‐sensitive lipase (HSL; E.C. 3.1.1.3), a multifunctional hydrolase that can mediate triacylglycerol cleavage in adipocytes.

Research Methods and Procedures: We performed constitutive gene targeting of the mouse HSL gene (Lipe), subsequently studied the adipose tissue phenotype clinically and histologically, and measured lipolysis in isolated adipocytes.

Results: Homozygous HSL^−/− mice have no detectable HSL peptide or cholesteryl esterase activity in adipose tissue, and heterozygous mice have intermediate levels with respect to wild‐type and deficient littermates. HSL‐deficient mice have normal body weight but reduced abdominal fat mass compared with normal littermates. Histologically, both white and brown adipose tissues in HSL^−/− mice show marked heterogeneity in cell size, with markedly enlarged adipocytes juxtaposed to cells of normal morphology. In isolated HSL^−/− adipocytes, lipolysis is not significantly increased by β₃‐adrenergic stimulation, but under basal conditions in the absence of added catecholamines, the lipolytic rate of isolated HSL^−/− adipocytes is at least as high as that of cells from normal controls. Cold tolerance during a 48‐hour period at 4 °C was similar in HSL^−/− mice and controls. Overnight fasting was well‐tolerated clinically by HSL^−/− mice, but after fasting, liver triglyceride content was significantly lower in HSL^−/− mice compared with wild‐type controls.

Conclusions: In isolated fat cells, the lipolytic rate after β‐adrenergic stimulation is mainly dependent on HSL. However, the observation of a normal rate of lipolysis in unstimulated HSL^−/− adipocytes suggests that HSL‐independent lipolytic pathway(s) exist in fat. Physiologically, HSL deficiency in mice has a modest effect under normal fed conditions and is compatible with normal maintenance of core body temperature during cold stress. However, the lipolytic response to overnight fasting is subnormal.