Hormone-Sensitive Lipase Knockouts
© Kraemer and Shen; licensee BioMed Central Ltd. 2006
Received: 30 January 2006
Accepted: 10 February 2006
Published: 10 February 2006
All treatments for obesity, including dietary restriction of carbohydrates, have a goal of reducing the storage of fat in adipocytes. The chief enzyme responsible for the mobilization of FFA from adipose tissue, i.e., lipolysis, is thought to be hormone-sensitive lipase (HSL). Studies of HSL knockouts have provided important insights into the functional significance of HSL and into adipose metabolism in general. Studies have provided evidence that HSL, though possessing triacylglycerol lipase activity, appears to be the rate-limiting enzyme for cholesteryl ester and diacylglycerol hydrolysis in adipose tissue and is essential for complete hormone stimulated lipolysis, but other triacylglycerol lipases are important in mediating triacylglycerol hydrolysis in lipolysis. HSL knockouts are resistant to both high fat diet-induced and genetic obesity, displaying reduced quantities of white with increased amounts of brown adipose tissue, increased numbers of adipose macrophages, and have multiple alterations in the expression of genes involved in adipose differentiation, including transcription factors, markers of adipocyte differentiation, and enzymes of fatty acid and triglyceride synthesis. With disruption of lipolysis by removal of HSL, there is a drastic reduction in lipogenesis and alteration in adipose metabolism.
Free fatty acids (FFA) are a major energy source for most tissues in mammals. Circulating FFA in plasma are primarily derived from adipose tissue, which is the main repository for the storage of triacylglycerol. All treatments for obesity, including dietary restriction of carbohydrates, have a goal of reducing the storage of fat in adipocytes. The chief enzyme responsible for the mobilization of FFA from adipose tissue, i.e., lipolysis, was thought to be hormone-sensitive lipase (HSL). This review addresses some aspects of the organization of components of fat metabolism in the adipocyte, lipolysis and lipogenesis, and how genetic manipulation of the pathway of lipolysis affects fat metabolism in the adipocyte.
Properties of HSL
HSL is an intracellular, neutral lipase that has broad substrate specificity, catalyzing the hydrolysis of triacylglycerol, diacylglycerol, monoacylglycerol, and cholesteryl esters, as well as retinyl esters; however, it possesses no phospholipase activity . Its activity against diacylglycerol is about 10-fold and 5-fold higher than its activity against triacylglycerol and monoacylglycerol, respectively, whereas its activity against cholesteryl esters is about twice its activity toward triacylglycerol. The hydrolytic activity of HSL against triacylglycerol and cholesteryl esters, but not against diacylglycerol, is stimulated by phosphorylation mediated primarily by protein kinase A (PKA) .
Studies of mice where HSL has been inactivated by homologous recombination have provided important insights into the functional significance of HSL and into adipose metabolism in general. HSL null mice have a normal physical appearance and are nonobese, lacking an apparent phenotype, with the exception that homozygous males have severe oligo- or azospermia and are infertile . However, careful examination of adipocytes does reveal abnormalities. There are histological changes in adipose tissue. Brown adipose tissue (BAT) displays increased cell size and white adipose tissue (WAT) displays an increased heterogeneity of cell size, with both larger and smaller cells being more prevalent [2, 3]. Examination of enzyme activity showed the complete absence of neutral cholesteryl ester hydrolase activity in adipose tissue, both WAT and BAT. Moreover, examination of other tissues where HSL is expressed, for instance adrenal, testis, ovary, and heart, showed the almost complete absence of neutral cholesteryl ester hydrolase activity [2, 4]. Thus, HSL is responsible for all, or practically all, of the neutral cholesteryl ester hydrolase activity in adipose and steroidogenic tissues. In contrast, triacylglycerol lipase activity in WAT was reduced by only 40–50% and triacylglycerol lipase activity in BAT was similar to wild type mice [2, 3, 5], suggesting the existence of one or more other neutral triacylglycerol lipases. Measurement of plasma concentrations of glycerol and FFA showed a 40–60% reduction in circulating levels under unstimulated conditions, whereas there was a small, but markedly attenuated, increase following isoproterenol injection, supporting an important, but not exclusive, role for HSL in lipolysis. In vitro studies of adipose cells isolated from HSL null mice recapitulated the in vivo findings for the most part; there was a marked defect or complete absence of catecholamine-stimulated glycerol release in adipose cells from HSL null mice, whereas catecholamine-stimulated FFA release was still observed, but attenuated [2, 3, 5, 6]. This apparent discrepancy in the release of glycerol and FFA from adipose cells of HSL null mice has been clarified by the observation that diacylglycerol content increased markedly in adipose tissue of HSL null mice . In both white and brown adipose tissue from HSL null mice, catecholamine-stimulation caused the release of small amounts of FFA without any stimulated glycerol release, and a marked accumulation of diacylglycerol . These results are consistent with the known substrate specificity of HSL being 10-fold higher against diacylglycerol than triacylglycerol. Indeed, direct measurement of diacylglycerol lipase activity showed that activity was reduced >95% in adipose tissue of HSL null mice, whereas diacylglycerol lipase activity was unaltered in muscle and liver . Therefore, HSL, though possessing triacylglycerol lipase activity, appears to be the rate-limiting enzyme for cholesteryl ester and diacylglycerol hydrolysis in adipose tissue and is essential for complete hormone stimulated lipolysis.
Lipases in adipocytes
The presence of residual triacylglycerol lipase activity in adipose tissue of HSL null mice has led to the search for other lipases responsible for the maintenance of the mobilization of FFA from fat. Using functional proteomics and oleic acid-linked agarose chromatography, Soni et al  identified carboxylesterase 3, also known as triacylglycerol hydrolase, as an abundant lipase in adipose tissue that continues to be expressed in HSL null mice. Using an in silico approach, Zimmermann et al  identified a protein that possessed lipase activity and was highly expressed in adipose tissue which they termed adipose triglyceride lipase (ATGL). ATGL had been identified by other authors, who termed it desnutrin  and calcium independent phospholipase A2ζ [10, 11]. ATGL contains a patatin-like sequence and has homology with several other patatin-like family members such as adiponutrin and GS2 or iPLA2η . Based on immunoprecipitation of ATGL from adipose tissue homogenates, ATGL was reported to account for 60–70% of the neutral triacylglycerol lipase activity in both wild-type and HSL null mice . However, it is apparent that there are multiple lipases in adipose tissue since 23 different proteins that potentially possess lipolytic or esterolytic activity in mouse adipose tissue have been identified using a proteomics approach that took advantage of fluorescent tags that mimic lipid substrates . In unpublished work we have recently examined the relative expression levels of 11 of the lipase/esterases in WAT using quantitative RT-PCR analysis. The proteins examined are: HSL, ATGL, adiponutrin, triacylglycerol hydrolase, α/β hydrolase containing mRNA 5 (also known as CGI-58) and 11 (also known as Beuren-Williams Syndrome Critical Region 21), acylpeptide hydrolase, esterase 1, esterase D, carboxylesterase ML1, and membrane-associated calcium-independent phospholipase A2γ. We found the following relative expression levels in normal mouse WAT in descending order of abundance: ATGL, HSL, triacylglycerol hydrolase, adiponutrin, membrane-associated calcium-independent phospholipase A2γ, esterase D, CGI-58, carboxylesterase ML1, acylpeptide hydrolase, Beuren-Williams Syndrome Critical Region 21, and esterase 1. Since the residual basal triacylglycerol lipase activity in WAT of HSL null mice indicates the existence of other lipase(s), we examined the expression levels of these other 10 lipase/esterases in WAT of HSL null mice. Surprisingly, almost all of the 10 lipase/esterases showed decreased levels of expression when compared with wild-type mice, with ATGL and triacylglycerol hydrolase reduced to only 30% of that of wild-type mice. Thus, multiple proteins with lipase/esterase activity are expressed in WAT and the absence of HSL does not result in a compensatory increase in the mRNA of any of these proteins, but rather in a global decrease in the expression of all lipases/esterases.
HSL KO mice are resistant to obesity
When the influence of HSL on the development of dietary induced obesity was examined by feeding high fat diets for 15 weeks, 20–26% lower weights were observed in male and female, respectively, HSL null mice compared with control . This occurred even though there was a higher food intake per body weight in HSL null mice and without any evidence of lipid malabsorption. There was increased fasting induced weight loss and higher core body temperatures in HSL null mice, consistent with an increase in energy expenditure. Even though total body weights were similar on a normal chow diet, total WAT mass was reduced in HSL null mice compared to control and this difference was exaggerated on a high fat diet, with total WAT weight more than 70% lower in HSL null mice than in control. In contrast to WAT, interscapular BAT was larger in HSL null mice than in control. In addition to resistance to diet-induced obesity, the absence of HSL also protects against genetic obesity, based on the observation that mice homozygous for both HSL and leptin deficiency (ob/ob) are 26% lighter than ob/ob mice and this is reflected by a reduction in WAT mass . The greater amount of heterogeneity of cell size in HSL null mice is exaggerated in mice with combined HSL and leptin deficiency, with a marked increase in small, lipid-devoid cells that appear to have characteristics of pre-adipocytes .
Altered adipose gene expression in HSL KO mice
In addition to accumulation of diacylglycerol in tissues noted above, cholesterol content in adipose tissues was increased in HSL null mice and this was accentuated by high fat feeding where cholesterol content was 5-fold higher in HSL null mice . Besides changes in histology and lipid content, HSL deficiency has multiple effects on adipose metabolism. Levels of mRNA expression of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα), two of the major transcription factors for adipogenesis [15, 16], were suppressed 40–70% in WAT of HSL null mice. Similarly, PPARα levels were also diminished ~70% in WAT of HSL null mice.
In parallel to the suppression of transcription factors, mRNA expression of markers of adipose differentiation such as adiponectin, leptin, resistin and adipsin, which are all humoral factors derived from adipose tissue, were decreased 60–90% . In contrast, expression of TNFα was up-regulated 2–3-fold in WAT and 5–10-fold in BAT of HSL null mice compared with control. Again consistent with the reduction in adipogenic transcription factors, all genes associated with fatty acid and triglyceride metabolism, such as adipocyte fatty acid binding protein, perilipin, lipoprotein lipase, glycerol-3-phosphate acyltransferase (GPAT), mitochondrial GPAT, acetyl-CoA carboxylase, fatty acid synthase, acyl-CoA synthetase, acyl-CoA:diacylglycerol acyltransferase 1 (DGAT-1), DGAT-2, ATP citrate lyase, were reduced in WAT of HSL null mice on either a normal chow or high fat diet [13, 17]. These alterations in gene expression resulted in a marked decrease in fatty acid esterification pathways and in the synthesis of neutral lipids and glycerol-phospholipids in WAT of HSL null mice . Thus, with disruption of lipolysis by removal of HSL, there is a drastic reduction in lipogenesis, which could allow a more efficient export of fatty acids released by lipolysis out of the cell.
In accordance with the down-regulation of adipogenic transcription factors, expression of insulin receptor, insulin receptor substrate-1 and glucose transporter 4 mRNA was decreased 30–80% in WAT of HSL null mice compared with control . However, examination of genes involved with cholesterol metabolism revealed that expression of 3-hydroxy-3-methylglutaryl-CoA (HMG CoA) reductase, the rate-limiting enzyme for cholesterol synthesis, was up-regulated ~2-fold; whereas, HMG CoA synthase-1, an enzyme upstream of HMG CoA reductase, was down-regulated 50–70% in WAT of HSL null mice . Surprisingly, expression of acyl-CoA:cholesterol acyltransferase 1, the enzyme that mediates the esterification of cholesterol to cellular cholesteryl esters was increased 2–4-fold in WAT and 5–8-fold in BAT. Expression levels of the lipogenic transcription factor sterol regulatory element binding protein-1c (SREBP-1c) were suppressed 50–75%, whereas expression of SREBP-2, which controls cholesterol synthesis and uptake, was up-regulated 2-fold. In parallel to the changes in SREBP-2, uncoupling protein 2 was increased 3–4-fold in BAT in HSL null mice .
Insulin and glucose metabolism in HSL KO mice
The effects of HSL deficiency on insulin and glucose metabolism is controversial in that several papers have been published that report conflicting results. For instance, overall insulin sensitivity in HSL null mice has been reported to be decreased by some authors  and normal by other authors [18, 19]. Hepatic insulin sensitivity has been reported to be increased [18, 19], whereas insulin sensitivity in adipose tissue and in muscle has been reported to be reduced [7, 17] or normal [18, 19]. Likewise, insulin secretion in HSL null mice has been reported to be either normal [7, 20] or reduced [21, 22]. The bases for these differences in results are not readily apparent, but may be due to differences in the genetic background on which the HSL null mice have been examined or may depend on whether the studies have been conducted in vitro or in vivo.
Macrophages in adipose tissue of HSL KO mice
Recent observations that there is increased macrophage infiltration into WAT in obesity have been extended to HSL null mice . Even though there is decreased WAT mass in HSL null mice as opposed to the increase seen in obesity, there is a greater prevalence of hypertrophied adipocytes in HSL null mice. Recent studies have observed that, in parallel to adipocyte hypertrophy, there is an increase in macrophages located in crown-like structures surrounding adipocytes with the macrophages scavenging adipocyte-free lipid droplets released from cells undergoing necrotic-like cell death . Thus, the greater numbers of macrophages in WAT in HSL null mice could contribute to the increase in TNFα and some of the other changes in adipocyte function observed.
List of abbreviations
Free fatty acids
protein kinase A
brown adipose tissue
white adipose tissue
adipose triglyceride lipase
peroxisome proliferators activated receptor
CCAAT/enhancer binding protein
tumor necrosis factorα
acyl-CoA: diacylglycerol acyltransferase
- HMG CoA:
sterol regulatory element binding protein
The authors thank previous members of the lab for their scientific contributions. This work was supported in part by a grant from the Research Service of the Department of Veterans Affairs.
- Kraemer FB, Shen WJ: Hormone-sensitive lipase: control of intracellular tri-(di-) acylglycerol and cholesteryl ester hydrolysis. J Lipid Res. 2002, 43: 1585-1594. 10.1194/jlr.R200009-JLR200.View ArticleGoogle Scholar
- Osuga J, Ishibashi S, Oka T, Yagyu H, Tozawa R, Fujimoto A, Shionoira F, Yahagi N, Kraemer FB, Tsutsumi O, Yamada N: Targeted disruption of hormone-sensitive lipase results in male sterility and adipocyte hypertrophy, but not in obesity. Proc Natl Acad Sci USA. 2000, 97: 787-792. 10.1073/pnas.97.2.787.View ArticleGoogle Scholar
- Wang SP, Laurin N, Himms-Hagen J, Rudnicki MA, Levy E, Robert MF, Pan L, Oligny L, Mitchell GA: The adipose tissue phenotype of hormone-sensitive lipase deficiency in mice. Obes Res. 2001, 9: 119-128.View ArticleGoogle Scholar
- Kraemer FB, Shen WJ, Natu V, Patel S, Osuga J, Ishibashi S, Azhar S: Adrenal neutral cholesteryl hydrolase: identification, subcellular distribution and sex differences. Endocrinology. 2002, 143: 801-806. 10.1210/en.143.3.801.View ArticleGoogle Scholar
- Haemmerle G, Zimmermann R, Hayn M, Theussl C, Waeg G, Wagner E, Sattler W, Magin TM, Wagner EF, Zechner R: Hormone-sensitive lipase deficiency in mice causes diglyceride accumulation in adipose tissue, muscle, and testis. J Biol Chem. 2002, 277: 4806-4815. 10.1074/jbc.M110355200.View ArticleGoogle Scholar
- Fortier M, Wang SP, Mauriege P, Semache M, Mfuma L, Li H, Levy E, Richard D, Mitchell GA: Hormone-sensitive lipase-independent adipocyte lipolysis during beta-adrenergic stimulation, fasting, and dietary fat loading. Am J Physiol Endocrinol Metab. 2004, 287: E282-288. 10.1152/ajpendo.00203.2003.View ArticleGoogle Scholar
- Mulder H, Sorhede-Winzell M, Contreras JA, Fex M, Strom K, Ploug T, Galbo H, Arner P, Lundberg C, Sundler F, Ahren B, Holm C: Hormone-sensitive lipase null mice exhibit signs of impaired insulin sensitivity whereas insulin secretion is intact. J Biol Chem. 2003, 278: 36380-36388. 10.1074/jbc.M213032200.View ArticleGoogle Scholar
- Soni KG, Lehner R, Metalnikov P, O'Donnell P, Semache M, Gao W, Ashman K, Pshezhetsky AV, Mitchell GA: Carboxylesterase 3 (EC 184.108.40.206) is a major adipocyte lipase. J Biol Chem. 2004, 279: 40683-40689. 10.1074/jbc.M400541200.View ArticleGoogle Scholar
- Zimmermann R, Strauss JG, Haemmerle G, Schoiswohl G, Birner-Gruenberger R, Riederer M, Lass A, Neuberger G, Eisenhaber F, Hermetter A, Zechner R: Fat mobilization in adipose tissue is promoted by adipose triglyceride lipase. Science. 2004, 306: 1383-1386. 10.1126/science.1100747.View ArticleGoogle Scholar
- Villena JA, Roy S, Sarkadi-Nagy E, Kim KH, Sul HS: Desnutrin, an adipocyte gene encoding a novel patatin domain-containing protein, is induced by fasting and glucocorticoids: ectopic expression of desnutrin increases triglyceride hydrolysis. J Biol Chem. 2004, 279: 47066-47075. 10.1074/jbc.M403855200.View ArticleGoogle Scholar
- Jenkins CM, Mancuso DJ, Yan W, Sims HF, Gibson B, Gross RW: Identification, cloning, expression, and purification of three novel human calcium-independent phospholipase A2 family members possessing triacylglycerol lipase and acylglycerol transacylase activities. J Biol Chem. 2004, 279: 48968-48975. 10.1074/jbc.M407841200.View ArticleGoogle Scholar
- Birner-Gruenberger R, Susani-Etzerodt H, Waldhuber M, Riesenhuber G, Schmidinger H, Rechberger G, Kollroser M, Strauss JG, Lass A, Zimmermann R, Haemmerle G, Zechner R, Hermetter A: The lipolytic proteome of mouse adipose tissue. Mol Cell Proteomics. 2005, 4: 1710-1717. 10.1074/mcp.M500062-MCP200.View ArticleGoogle Scholar
- Harada K, Shen WJ, Patel S, Natu V, Wang J, Osuga J, Ishibashi S, Kraemer FB: Resistance to high-fat diet-induced obesity and altered expression of adipose-specific genes in HSL-deficient mice. Am J Physiol Endocrinol Metab. 2003, 285: E1182-1195.View ArticleGoogle Scholar
- Sekiya M, Osuga JI, Okazaki H, Yahagi N, Harada K, Shen WJ, Tamura Y, Tomita S, Iizuka Y, Ohashi K, Okazaki M, Sata M, Nagai R, Fujita T, Shimano H, Kraemer FB, Yamada N, Ishibashi S: Absence of hormone-sensitive lipase inhibits obesity and adipogenesis in Lepob/ob mice. J Biol Chem. 2004, 279: 15084-15090. 10.1074/jbc.M310985200.View ArticleGoogle Scholar
- Wu Z, Rosen ED, Brun R, Hauser S, Adelmant G, Troy AE, McKeon C, Darlington GJ, Spiegelman BM: Cross-regulation of C/EBP alpha and PPAR gamma controls the transcriptional pathway of adipogenesis and insulin sensitivity. Mol Cell. 1999, 3: 151-158. 10.1016/S1097-2765(00)80306-8.View ArticleGoogle Scholar
- Rosen ED, Hsu CH, Wang X, Sakai S, Freeman MW, Gonzalez FJ, Spiegelman BM: C/EBPalpha induces adipogenesis through PPARgamma: a unified pathway. Genes Dev. 2002, 16: 22-26. 10.1101/gad.948702.View ArticleGoogle Scholar
- Zimmermann R, Haemmerle G, Wagner EM, Strauss JG, Kratky D, Zechner R: Decreased fatty acid esterification compensates for the reduced lipolytic activity in hormone-sensitive lipase-deficient white adipose tissue. J Lipid Res. 2003, 44: 2089-2099. 10.1194/jlr.M300190-JLR200.View ArticleGoogle Scholar
- Voshol PJ, Haemmerle G, Ouwens DM, Zimmermann R, Zechner R, Teusink B, Maassen JA, Havekes LM, Romijn JA: Increased hepatic insulin sensitivity together with decreased hepatic triglyceride stores in hormone-sensitive lipase-deficient mice. Endocrinology. 2003, 144: 3456-3462. 10.1210/en.2002-0036.View ArticleGoogle Scholar
- Park SY, Kim HJ, Wang S, Higashimori T, Dong J, Kim YJ, Cline G, Li H, Prentki M, Shulman GI, Mitchell GA, Kim JK: Hormone-sensitive lipase knockout mice have increased hepatic insulin sensitivity and are protected from short-term diet-induced insulin resistance in skeletal muscle and heart. Am J Physiol Endocrinol Metab. 2005, 289: E30-39. 10.1152/ajpendo.00251.2004.View ArticleGoogle Scholar
- Fex M, Olofsson CS, Fransson U, Bacos K, Lindvall H, Sorhede-Winzell M, Rorsman P, Holm C, Mulder H: Hormone-sensitive lipase deficiency in mouse islets abolishes neutral cholesterol ester hydrolase activity but leaves lipolysis, acylglycerides, fat oxidation, and insulin secretion intact. Endocrinology. 2004, 145: 3746-3753. 10.1210/en.2003-1673.View ArticleGoogle Scholar
- Roduit R, Masiello P, Wang SP, Li H, Mitchell GA, Prentki M: A role for hormone-sensitive lipase in glucose-stimulated insulin secretion: a study in hormone-sensitive lipase-deficient mice. Diabetes. 2001, 50: 1970-1975.View ArticleGoogle Scholar
- Peyot ML, Nolan CJ, Soni K, Joly E, Lussier R, Corkey BE, Wang SP, Mitchell GA, Prentki M: Hormone-sensitive lipase has a role in lipid signaling for insulin secretion but is nonessential for the incretin action of glucagon-like peptide 1. Diabetes. 2004, 53: 1733-1742.View ArticleGoogle Scholar
- Cinti S, Mitchell G, Barbatelli G, Murano I, Ceresi E, Faloia E, Wang S, Fortier M, Greenberg AS, Obin MS: Adipocyte death defines macrophage localization and function in adipose tissue of obese mice and humans. J Lipid Res. 2005, 46: 2347-2355. 10.1194/jlr.M500294-JLR200.View ArticleGoogle Scholar
- Le Lay S, Krief S, Farnier C, Lefrère I, Le Liepvre X, Bazin R, Ferré P, Dugail I: Cholesterol, a cell size-dependent signal that regulates glucose metabolism and gene expression in adipocytes. J Biol Chem. 2001, 276: 16904-16910. 10.1074/jbc.M010955200.View ArticleGoogle Scholar
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