HSL (hormone-sensitive lipase) is a key enzyme in the mobilization of fatty acids from acylglycerols in adipocytes as well as non-adipocytes. In adipocytes, catecholamines stimulate lipolysis mainly through PKA (protein kinase A)-mediated phosphorylation of HSL and perilipin, a protein coating the lipid droplet. The anti-lipolytic action of insulin is mediated mainly via lowered cAMP levels, accomplished through activation of phosphodiesterase 3B. Phosphorylation of HSL by PKA occurs at three sites, the serines 563, 659 and 660, both in vitro and in primary rat adipocytes. Phosphorylation of Ser-659 and -660 is required for in vitro activation as well as translocation from the cytosol to the lipid droplet, whereas the role of the third PKA site remains elusive. Adipocytes isolated from homozygous HSL-null mice, generated in our laboratory, exhibit completely blunted catecholamine-induced glycerol release and reduced fatty acid release, suggesting the presence of additional, although not necessarily hormone-activatable, triacylglycerol lipase(s). Basal hyperinsulinaemia, release of exaggerated amounts of insulin during glucose challenges and retarded glucose disposal during insulin tolerance tests suggest that HSL-null mice are insulin resistant. Liver, adipose tissue and skeletal muscle appear all to be sites of impaired insulin sensitivity in HSL-null mice.

Monoglyceride lipase catalyzes the last step in the hydrolysis of stored triglycerides in the adipocyte and presumably also complements the action of lipoprotein lipase in degrading triglycerides from chylomicrons and very low density lipoproteins. Monoglyceride lipase was cloned from a mouse adipocyte cDNA library. The predicted amino acid sequence consisted of 302 amino acids, corresponding to a molecular weight of 33,218. The sequence showed no extensive homology to other known mammalian proteins, but a number of microbial proteins, including two bacterial lysophospholipases and a family of haloperoxidases, were found to be distantly related to this enzyme. By means of multiple sequence alignment and secondary structure prediction, the structural elements in monoglyceride lipase, as well as the putative catalytic triad, were identified. The residues of the proposed triad, Ser-122, in a GXSXG motif, Asp-239, and His-269, were confirmed by site-directed mutagenesis experiments. Northern blot analysis revealed that monoglyceride lipase is ubiquitously expressed among tissues, with a transcript size of about 4 kilobases.The sequential hydrolysis of stored triglycerides in adipose tissue is the result of a combined action of two lipases, hormone-sensitive lipase and monoglyceride lipase (MGL 1 ; EC 3.1.1.23). Hormone-sensitive lipase catalyzes the first and ratelimiting step, the hydrolysis of triglycerides, and also the subsequent hydrolysis of di-and monoglycerides (1). Hormonesensitive lipase has a marked, although not absolute, preference for the primary ester bond of glyceride substrates. It has been shown that MGL is required to obtain a complete degradation of monoglycerides to fatty acids and glycerol, i.e. in the absence of MGL there is an accumulation of monoglycerides (mainly 2-monoglycerides) (2). The main physiological role for MGL is probably to assure complete hydrolysis of monoglycerides formed during the lipolysis of stored triglycerides of the adipocyte. Another role for the enzyme could be to catalyze the hydrolysis of 2-monoglycerides formed as a result of lipoprotein lipase-catalyzed hydrolysis of triglycerides from chylomicrons and very low density lipoproteins. Lipoprotein lipase has monoglyceride-hydrolyzing activity, with an absolute preference for the primary ester bond (3). This lipase could therefore catalyze the hydrolysis of 1(3)-monoglycerides, which are formed through isomerization from 2-monoglycerides. However, since the rate of isomerization at pH 7.4 is low, it is more likely that a substantial fraction of the 2-monoglycerides, formed through the action of lipoprotein lipase, is transported into the adipocyte and hydrolyzed by MGL (4). It should be pointed out that besides these two enzymes, there is no evidence for any other monoglyceride-hydrolyzing activity of adipose tissue.MGL has been extensively purified from rat adipose tissue in our laboratory (5). The limited amounts of purified enzyme obtained have been used to study some of its enzymological and biochemical propertie...

Hormone-sensitive lipase (HSL) is the rate-limiting enzyme in lipolysis. Stimulation of rat adipocytes with isoproterenol results in phosphorylation of HSL and a 50-fold increase in the rate of lipolysis. In this study, we used site-directed mutagenesis and two-dimensional phosphopeptide mapping to show that phosphorylation sites other than the previously identified Ser-563 are phosphorylated in HSL in response to isoproterenol stimulation of 32 P-labeled rat adipocytes. Phosphorylation of HSL in adipocytes in response to isoproterenol and in vitro phosphorylation of HSL containing Ser 3 Ala mutations in residues 563 and 565 (S563A,S565A) with protein kinase A (PKA), followed by tryptic phosphopeptide mapping resulted in two tryptic phosphopeptides. These tryptic phosphopeptides co-migrated with the phosphopeptides released by the same treatment of F 654 HPRRSSQGVLHMPLYSSPIVK 675 phosphorylated with PKA. Analysis of the phosphorylation site mutants, S659A, S660A, and S659A,S660A disclosed that mutagenesis of both Ser-659 and Ser-660 was necessary to abolish the activation of HSL toward a triolein substrate after phosphorylation with PKA. Mutation of Ser-563 to alanine did not cause significant change of activation compared with wild-type HSL. Hence, our results demonstrate that in addition to the previously identified Ser-563, two other PKA phosphorylation sites, Ser-659 and Ser-660, are present in HSL and, furthermore, that Ser-659 and Ser-660 are the major activity controlling sites in vitro.Free fatty acids stored as triacylglycerols in the adipocytes comprise the quantitatively most important energy substrate in mammals. Hormone-sensitive lipase (HSL) 1 (EC 3.1.1.3) catalyzes the first and rate-limiting step in the hydrolysis of stored triacylglycerols and is thereby a key enzyme in the mobilization of free fatty acids from adipose tissue (1). The hormonal and neural control of lipolysis is exerted by regulation of HSL activity, mediated by reversible phosphorylation (2, 3). In response to catecholamines and other fast-acting lipolytic hormones, HSL is activated through the phosphorylation by protein kinase A (PKA). The major antilipolytic hormone insulin prevents cAMP-mediated phosphorylation and activation of HSL (3). The antilipolytic effect of insulin is brought about mainly by activation of phosphodiesterase 3B (4).HSL has in intact rat adipocytes been reported to be phosphorylated at two sites (3). These sites were later identified as Ser-563 (5) and Ser-565 (6). In hormonally quiescent cells, only Ser-565 is phosphorylated (3). Stimulation with noradrenaline increases the phosphorylation extent of Ser-563 to that of Ser-565 (3). Ser-563 is also phosphorylated in vitro by PKA (5). Ser-565 has been shown to be phosphorylated in vitro by the 5Ј-AMP-activated kinase (6). This kinase, which phosphorylates and regulates the activity of other key enzymes in lipid metabolism (7), acetyl-CoA carboxylase (fatty acid synthesis), and 3-hydroxy-3-methylglutaryl-CoA (cholesterol synthesis), has also been suggested ...

The mobilization of fat stored in adipose tissue is mediated by hormone-sensitive lipase (HSL) and the recently characterized adipose triglyceride lipase (ATGL), yet their relative importance in lipolysis is unknown. We show that a novel potent inhibitor of HSL does not inhibit other lipases. The compound counteracted catecholamine-stimulated lipolysis in mouse adipocytes and had no effect on residual triglyceride hydrolysis and lipolysis in HSL-null mice. In human adipocytes, catecholamine-and natriuretic peptide-induced lipolysis were completely blunted by the HSL inhibitor. When fat cells were not stimulated, glycerol but not fatty acid release was inhibited. HSL and ATGL mRNA levels increased concomitantly during adipocyte differentiation. Abundance of the two transcripts in human adipose tissue was highly correlated in habitual dietary conditions and during a hypocaloric diet, suggesting common regulatory mechanisms for the two genes. Comparison of obese and nonobese subjects showed that obesity was associated with a decrease in catecholamine-induced lipolysis and HSL expression in mature fat cells and in differentiated preadipocytes. In conclusion, HSL is the major lipase for catecholamine-and natriuretic peptide-stimulated lipolysis, whereas ATGL mediates the hydrolysis of triglycerides during basal lipolysis. Decreased catecholamine-induced lipolysis and low HSL expression constitute a possibly primary defect in obesity. Diabetes 54:3190 -3197, 2005 O besity, which is characterized by an excess of fat stores, is the most important risk factor for type 2 diabetes. Adipose tissue lipolysis leads to the hydrolysis of triglycerides and release of free fatty acids (FFAs). Because of the link between elevated circulating FFA levels and the development of insulin resistance and the metabolic syndrome (1,2), adipose tissue lipolysis constitutes a target for the drug industry. Nicotinic acid, which acts by inhibiting adipose tissue lipolysis, was the first extensively used lipid-lowering agent (3). Catecholamines and natriuretic peptides are the major hormones stimulating this catabolic pathway in humans (4). Resistance to catecholamine-induced lipolysis in subcutaneous adipose tissue has been demonstrated in obese adults and children (5,6) and is attributed to decreased expression of lipolytic ␤ 2 -adrenoceptors (7), increased antilipolytic properties of ␣ 2 -adrenoceptors (8), and decreased expression of hormone-sensitive lipase (HSL) (9). It is possible that the HSL defect is the most important factor because it is also observed in nonobese first-degree relatives to obese subjects (10) and because there is a positive relationship between lipolytic capacity and HSL expression in human subcutaneous fat cells (11).The rate-limiting role of HSL in adipose tissue lipolysis has been challenged by the data from HSL knockout mice (12-15). Catecholamine-induced lipolysis is abrogated, but residual basal lipolysis is observed in adipocytes from HSL-null mice. These data suggest the existence of non-HSL lipases in adipos...

Improved continuous acoustic particle separation (separation efficiency close to 100%) and separation of erythrocytes (red blood cells) from lipid microemboli in whole blood is reported.