Abstract
Hormone-sensitive lipase (HSL) is expressed predominantly in white and brown adipose tissue where it is believed to play a crucial role in the lipolysis of stored triglycerides (TG), thereby providing the body with energy substrate in the form of free fatty acids (FFA). From in vitro assays, HSL is known to hydrolyze TG, diglycerides (DG), cholesteryl esters, and retinyl esters. In the current study we have generated HSL knock-out mice and demonstrate three lines of evidence that HSL is instrumental in the catabolism of DG in vivo. First, HSL deficiency in mice causes the accumulation of DG in white adipose tissue, brown adipose tissue, skeletal muscle, cardiac muscle, and testis. Second, when tissue extracts were used in an in vitro lipase assay, a reduced FFA release and the accumulation of DG was observed in HSL knock-out mice which did not occur when tissue extracts from control mice were used. Third, in vitro lipolysis experiments with HSL-deficient fat pads demonstrated that the isoproterenol-stimulated release of FFA was decreased and DG accumulated intracellularly resulting in the essential absence of the isoproterenol-stimulated glycerol formation typically observed in control fat pads. Additionally, the absence of HSL in white adipose tissue caused a shift of the fatty acid composition of the TG moiety toward increased long chain fatty acids implying a substrate specificity of the enzyme in vivo. From these in vivo results we conclude that HSL is the rate-limiting enzyme for the cellular catabolism of DG in adipose tissue and muscle.
Highlights
Hormone-sensitive lipase (HSL) is expressed predominantly in white and brown adipose tissue where it is believed to play a crucial role in the lipolysis of stored triglycerides (TG), thereby providing the body with energy substrate in the form of free fatty acids (FFA)
The linearized targeting vector was electroporated in HM-1 embryonic stem (ES) cells [30], and homologous recombination events were confirmed by antibiotic resistance screening in cell cultures and Southern blot analysis of ES cell genomic DNA
In vitro studies have shown that in addition to TG, HSL catalyzes the hydrolysis of DG, MG [37, 38], cholesteryl esters (CE) [39, 40], and retinyl esters [25]
Summary
Clones that exhibited homologous recombination at the 5Ј short arm of the targeting vector and 3Ј to the third loxP site (Fig. 1B) were expanded and electroporated with 100 g of circular pCrePac (kindly provided by Dr Takeshi Yagi, Japan) for Cre-mediated deletion (Fig. 2A) of the NEO HSV-TK cassette [31]. If the loxP sequences 1 and 3 were used for recombination (Fig. 2A, Type I deletion), exons 2–7 including the NEO HSV-TK cassette were deleted (complete HSL knock-out allele). The targeting vector contains 8.4-kb homologous HSL genomic DNA, with 1.7 kb located 5Ј and 6.7 kb located 3Ј of the loxP-flanked NEO HSV-TK cassette (loxP sites are indicated by triangles). FA Composition—For analysis of FFA and TG-associated FA in epididymal fat pads, tissue specimens were weighed, and the lipids were extracted with 2 ml of H2O and 4 ml of chloroform/methanol (2:1) for 40 min at room temperature. TG and DG corresponding bands were scraped from the plates and quantitated as described above
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