Human Hormone-Sensitive Lipase: Expression and Large-Scale Purification from a Baculovirus/Insect Cell System

Contreras, Juan Antonio; Danielsson, Birgitta; Johansson, Camilla; Østerlund, Torben; Langin, Dominique; Holm, Cecilia

doi:10.1006/prep.1997.0821

Cited by 28 publications

(29 citation statements)

References 18 publications

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“…The antiserum was shown to specifically recognize full-length human HSL (HSL-long), whereas it did not recognize the splice variant lacking exon 6 (HSL-short). Antibodies recognizing both HSL-long and -short were generated in chicken as previously described (26,27). In short, immunized chicken antiserum was affinity-purified against recombinant rat HSL coupled to a CNBr-activate Sepharose 4B column (Amersham Pharmacia Biotech, Uppsala, Sweden).…”

Section: Methodsmentioning

confidence: 99%

A Unique Defect in the Regulation of Visceral Fat Cell Lipolysis in the Polycystic Ovary Syndrome as an Early Link to Insulin Resistance

et al. 2002

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The etiology of polycystic ovary syndrome (PCOS) is unknown. However, PCOS has a strong resemblance to the insulin resistance (metabolic) syndrome, where an increased rate of visceral fat cell lipolysis is believed to play a pathophysiological role. We hypothesized that primary defects in visceral lipolysis might also exist in PCOS. Ten young, nonobese, and otherwise healthy PCOS women were compared with 13 matched control women. In vitro lipolysis regulation and stoichiometric properties of the final step in lipolysis activation, namely the protein kinase A (PKA)-hormone sensitive lipase (HSL) complex, were investigated in isolated visceral (i.e., omental) fat cells. Body fat distribution and circulating levels of insulin, glucose, and lipids were normal in PCOS women. However, in vivo insulin sensitivity was slightly decreased (P ‫؍‬ 0.03). Catecholamineinduced adipocyte lipolysis was markedly (i.e., about twofold) increased in PCOS women due to changes at the postreceptor level, although there was no change in the antilipolytic properties of visceral fat cells. Western blot analyses of visceral adipose tissue showed twofold increased levels of the catalytic and the regulatory I␣ components of PKA. In contrast, the regulatory RII␤ component of PKA was almost 50% decreased in visceral adipose tissue in PCOS women. Recent studies on genetically modified mice have shown that a similar transition in the regulatory PKA units induces an increased lipolytic response to catecholamines. Further analysis showed that the level of HSL-short, an enzymatically inactive splice form of HSL, was decreased in PCOS (P < 0.01). The altered lipolysis in PCOS is different from that observed in visceral fat cells in the insulin resistance syndrome that occurs at the level of adrenergic receptors. We concluded that increased catecholamine-induced lipolysis in visceral fat cells may be due to unique alterations in the stoichiometric properties of the adipose PKA-HSL holoenzymes. This could be an early and possibly primary lipolysis defect in PCOS.

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Section: Methodsmentioning

confidence: 99%

A Unique Defect in the Regulation of Visceral Fat Cell Lipolysis in the Polycystic Ovary Syndrome as an Early Link to Insulin Resistance

et al. 2002

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“…Activation of HSL requires phosphorylation by PKA or PKG on specific regulatory serine residues. In rat HSL, these sites are Ser 563 , Ser 659 , and Ser 660 (Stralfors et al 1984, Garton et al 1988, Anthonsen et al 1998 corresponding to the human residues Ser 552 , Ser 649 , and Ser 650 (Contreras et al 1998, Watt et al 2006. The functional role of these sites is different; phosphorylation of Ser 563 is thought to promote the translocation of HSL from the cytosol to LDs (Daval et al 2005), while phosphorylation of Ser 659 and Ser 660 is critical for activation of the intrinsic enzymatic activity (Anthonsen et al 1998 (Fig.…”

Section: Hormone-sensitive Lipasementioning

confidence: 99%

Dissecting adipose tissue lipolysis: molecular regulation and implications for metabolic disease

Nielsen¹,

Jessen²,

Jørgensen³

et al. 2014

Journal of Molecular Endocrinology

300

239

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Lipolysis is the process by which triglycerides (TGs) are hydrolyzed to free fatty acids (FFAs) and glycerol. In adipocytes, this is achieved by sequential action of adipose TG lipase (ATGL), hormone-sensitive lipase (HSL), and monoglyceride lipase. The activity in the lipolytic pathway is tightly regulated by hormonal and nutritional factors. Under conditions of negative energy balance such as fasting and exercise, stimulation of lipolysis results in a profound increase in FFA release from adipose tissue (AT). This response is crucial in order to provide the organism with a sufficient supply of substrate for oxidative metabolism. However, failure to efficiently suppress lipolysis when FFA demands are low can have serious metabolic consequences and is believed to be a key mechanism in the development of type 2 diabetes in obesity. As the discovery of ATGL in 2004, substantial progress has been made in the delineation of the remarkable complexity of the regulatory network controlling adipocyte lipolysis. Notably, regulatory mechanisms have been identified on multiple levels of the lipolytic pathway, including gene transcription and translation, post-translational modifications, intracellular localization, protein-protein interactions, and protein stability/ degradation. Here, we provide an overview of the recent advances in the field of AT lipolysis with particular focus on the molecular regulation of the two main lipases, ATGL and HSL, and the intracellular and extracellular signals affecting their activity.

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“…The substrate for the measurement of triglyceride hydrolase activity, containing triolein and [9,10-3 H]triolein (PerkinElmer Life Sciences), was emulsified with phosphatidylcholine/phosphatidylinositol (3:1) using a probe sonicator (Virsonic 475, Virtis, Gardiner, NJ), as described previously (31). Cell lysates (0.1 ml) from cells expressing the constructs described in supplemental Table 1 were added to 0.1 ml of the substrate and then incubated in a water bath at 37°C for 30 min.…”

Section: Assay For Triglyceride Hydrolase Activities Of Cell Extracts-mentioning

confidence: 99%

“…The reaction was terminated by adding 3.25 ml of methanol/chloroform/heptane (10:9:7) and 1 ml of 0.1 M potassium carbonate, 0.1 M boric acid, pH 10.5. After centrifugation at 800 ϫ g for 20 min, the radioactivity in 0.5 ml of the upper phase was determined by liquid scintillation counting, as described previously (31).…”

Section: Assay For Triglyceride Hydrolase Activities Of Cell Extracts-mentioning

confidence: 99%

Activation of Hormone-sensitive Lipase Requires Two Steps, Protein Phosphorylation and Binding to the PAT-1 Domain of Lipid Droplet Coat Proteins

Wang

Dalen

et al. 2009

Journal of Biological Chemistry

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146

159

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Lipid droplets are cellular organelles, structurally similar to lipoprotein particles. Lipid droplets include a neutral lipid core composed largely of triglycerides, surrounded by a phospholipid monolayer and coated with surface proteins that provide an interface for various aspects of lipid metabolism, including lipid transport, lipogenesis, and lipolysis (1-5). Lipolysis is an important mechanism by which cells release energy stored in lipid droplets; its impairment has been linked to cellular lipotoxicity and insulin resistance (6). Studies are needed to gain an understanding of the underlying molecular mechanisms regulating lipolysis. Although all cells are equipped to perform lipolysis, the extent of lipid accumulation and specific components of the lipolytic pathway are variable, depending on the type of cell.Numerous recent studies have led to consensus that members of the PAT family of proteins, originally named for Perilipin, Adipose differentiation-related protein (ADFP) 4 and Tail Interacting Protein 47 (TIP47), play conserved structural and functional roles on lipid droplets (6 -9). Proteomic studies have identified a "signature" composition for lipid droplets from a variety of types of cells that includes at least one PAT family member. In mammalian cells, the PAT family includes perilipin

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Human Hormone-Sensitive Lipase: Expression and Large-Scale Purification from a Baculovirus/Insect Cell System

Cited by 28 publications

References 18 publications

A Unique Defect in the Regulation of Visceral Fat Cell Lipolysis in the Polycystic Ovary Syndrome as an Early Link to Insulin Resistance

A Unique Defect in the Regulation of Visceral Fat Cell Lipolysis in the Polycystic Ovary Syndrome as an Early Link to Insulin Resistance

Dissecting adipose tissue lipolysis: molecular regulation and implications for metabolic disease

Activation of Hormone-sensitive Lipase Requires Two Steps, Protein Phosphorylation and Binding to the PAT-1 Domain of Lipid Droplet Coat Proteins

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