Measurement of pancreatic lipase activity in serum by a kinetic                colorimetric assay using a new chromogenic substrate

Panteghini, Mauro; Bonora, R; Pagani, F.

doi:10.1258/0004563011900876

Cited by 90 publications

(76 citation statements)

References 17 publications

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“…Lipoprotein Lipase Activity Assays-In vitro LPL activity assays were carried out using 1,2-O-dilauryl-rac-glycero-3-glutaric acid-(6Ј-methylresorufin) ester (DGGR; Sigma) as a fluorescent substrate (32). Purified bovine milk LPL (Sigma) or human recombinant LPL was used.…”

Section: Methodsmentioning

confidence: 99%

The Angiopoietin-like Proteins ANGPTL3 and ANGPTL4 Inhibit Lipoprotein Lipase Activity through Distinct Mechanisms

Shan

Liu

et al. 2009

Journal of Biological Chemistry

127

112

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Two members of the angiopoietin-like family of proteins, ANGPTL3 and ANGPTL4, have been shown to play important roles in modulating lipoprotein metabolism in the body. Both proteins were found to suppress lipoprotein lipase (LPL) activity in vitro as well as in vivo. However, their mechanisms of inhibition remained poorly understood. Using enzyme kinetic analysis with purified recombinant proteins, we have found key mechanistic differences between ANGPTL3 and ANGPTL4. ANGPTL3 reduced LPL catalytic activity but did not significantly alter its self-inactivation rate. In contrast, ANGPTL4 suppressed LPL by accelerating the irreversible inactivation of LPL. Furthermore, heparin was able to overcome the inhibitory effect of ANGPTL3 on LPL but not that of ANGPTL4. Site-directed mutagenesis demonstrated the critical function of Glu 40 in ANGPTL4. In contrast, when cysteine residues involved in disulfide bond formation were mutated to serines, ANGPTL4 retained its activity. Taken together, our data provide a more detailed view of the structure and mechanisms of these proteins. The finding that ANGPTL3 and ANGPTL4 inhibit LPL activity through distinct mechanisms indicates that the two proteins play unique roles in modulation of lipid metabolism in vivo. Lipoprotein lipase (LPL)2 is an essential enzyme that catalyzes the hydrolysis of triglycerides to generate free fatty acids and monoacylglycerol (for review, see Refs. 1 and 2). It is synthesized and secreted by adipocytes, macrophages, and muscle cells and then bound to the vascular endothelium by heparin sulfate proteoglycans and GPIHBP1 (glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1), a recently discovered protein (3, 4). LPL anchored as such releases free fatty acids and monoacylglycerol from triglycerides carried by chylomicron and very low density lipoprotein particles (5-9) and thus plays a major role in lipid metabolism. LPL-deficient subjects have severe hypertriglyceridemia and increased risk of arteriosclerosis (10). In contrast, subjects with slightly increased LPL activity were found to have lower triglyceride levels and decreased risk of cardiovascular diseases (11).It is well known that LPL is rapidly inactivated in vivo, but the underlying mechanism is unknown (12, 13). Recently, two secreted proteins were found to inhibit LPL activity both in vitro and in vivo. These two proteins, known as ANGPTL3 and ANGPTL4, are members of the angiopoietin-like protein family (14 -17). They share 31% overall sequence homology, with an N-terminal domain containing a coiled-coil region and a C-terminal fibrinogen-like domain that is cleaved off in vivo (16,18). Both proteins are found to inhibit LPL activity in vitro (16,18). Overexpression of ANGPTL3 and ANGTPL4 in mice led to extremely high blood levels of triglycerides and cholesterol (15, 19 -21). Knock-out of either gene in mice results in much lower blood levels of these lipids (14,(17)(18)(19)(22)(23)(24). Furthermore, post-heparin plasma LPL activity is significantly elevated in...

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Section: Methodsmentioning

confidence: 99%

The Angiopoietin-like Proteins ANGPTL3 and ANGPTL4 Inhibit Lipoprotein Lipase Activity through Distinct Mechanisms

Shan

Liu

et al. 2009

Journal of Biological Chemistry

127

112

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“…The ITC assay is an alternative to assays that use radiolabeled (40) or fluorogenic substrates (41,42), or directly measure released fatty acids by NEFA kits (43). Because plasma contains free fatty acids at high concentrations, [0.3-1 mM (44)], the NEFA kit is applicable only when significant amounts of fatty acids have been produced by the added LPL.…”

Section: Figmentioning

confidence: 99%

Lipoprotein lipase activity and interactions studied in human plasma by isothermal titration calorimetry

Reimund

Kovrov

Olivecrona

et al. 2017

Journal of Lipid Research

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LPL is produced and secreted from parenchymal cells like adipocytes and myocytes for transport to the luminal side of the endothelium via interaction with the glycosylphosphatidylinositol-anchored high density lipoprotein binding protein 1 (GPIHBP1) (8). Several plasma components have been shown to directly or indirectly modulate the activity of LPL. apoC-II and apoA-V increase the activity of LPL, while apoC-I, apoC-III, angiopoietin-like protein (ANGPTL)3, ANGPTL4, and ANGPTL8 decrease the activity (1, 9). The expression of each of these proteins depends on nutritional and hormonal factors, so that lipid uptake in tissues to a large extent is regulated by posttranslational effects on LPL (1, 9). It is possible that the macromolecular environment in plasma itself may be an influence on the interaction of LPL with its ligands. The protein concentration of plasma (80 g/l) has been shown to cause significant crowding effects (10). It is also possible that some plasma regulators of LPL activity have not been identified yet.LPL activity can be measured in vitro by using artificial, usually emulsified, systems of radiolabeled, fluorogenic, or chromogenic substrates, or isolated triglyceride-rich lipoproteins (TRLs). The reaction products are detected at certain time points by chemical quantification or by determination of radioactivity or fluorescence. These methods have been used to unravel important aspects of the action of LPL and also to quantitate the levels of LPL activity in cells and tissues. Only small amounts of LPL activity are normally present in the circulating blood (11). Therefore, intravenous injections of heparin are made to release LPL from its endothelial binding sites. Determination of LPL activity in postheparin plasma, using artificial substrate systems, is considered to give an estimation of the amount of active LPL at the vascular endothelium (12).Lack of a suitable technique for continuous monitoring of triglyceride hydrolysis in plasma has hampered the understanding of the action of LPL under physiological Abstract LPL hydrolyzes triglycerides in plasma lipoproteins. Due to the complex regulation mechanism, it has been difficult to mimic the physiological conditions under which LPL acts in vitro. We demonstrate that isothermal titration calorimetry (ITC), using human plasma as substrate, overcomes several limitations of previously used techniques. The high sensitivity of ITC allows continuous recording of the heat released during hydrolysis. Both initial rates and kinetics for complete hydrolysis of plasma lipids can be studied. The hydrolytic breakdown of plasma triglycerides by LPL at the capillary endothelium is a crucial event that contributes to control of the levels of triglycerides in plasma (1, 2). Many recent studies support the view that an elevated level of triglycerides in plasma is an independent risk factor for development of atherosclerosis (3-5). Therefore, the LPL system is considered to be an interesting target for drug design (6, 7). Education and Research Grant IUT 19-9,...

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“…Lipase activity was evaluated as reported previously using the method of Panteghini et al (2001) based on the use of 1,2-odilauryl-rac-glycero-3-glutaric acid-(6 0 -methylresorufin) ester (DGGR) as substrate. Sperm extracts weighing 50 mg were loaded into individual cuvettes containing a buffer for spectrophotometric determination (Panteghini et al 2001).…”

Section: Lipase Activity Assaymentioning

confidence: 99%

“…Sperm extracts weighing 50 mg were loaded into individual cuvettes containing a buffer for spectrophotometric determination (Panteghini et al 2001). DGGR is cleaved by lipase, resulting in an unstable dicarbonic acid ester, which is spontaneously hydrolyzed to yield glutaric acid and methylresorufin, a bluish-purple chromophore with peak absorption at 580 nm.…”

Section: Lipase Activity Assaymentioning

confidence: 99%

Human sperm anatomy and endocrinology in varicocele: role of androgen receptor

Guido¹,

Santoro²,

Amicis³

et al. 2014

Reproduction

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The study of androgens involved in male reproduction has been object of intense efforts, while their reported action on human male gametes is limited. We previously described the presence of androgen receptor (AR) in sperm with a role related to the modulation of the PI3K pathway. In the present study, we investigated the expression of AR and its ultrastructural location in normal sperm as well as in spermatozoa obtained from varicocele patients. We observed a reduced AR content in varicocele sperm with respect to healthy sperm by western blot analysis and transmission electron microscopy (TEM). The ultrastructural location of AR was detected mainly on the head membrane as well as in the nucleus, neck, and mitochondria. Influence of dihydrotestosterone (DHT) treatment on cholesterol efflux was increased in normal sperm, while it was reduced or absent in varicocele sperm. To better understand DHT/AR significance in human male gametes, we evaluated triglyceride content and lipase, acyl-CoA dehydrogenase, and glucose-6-phosphate dehydrogenase activities upon DHT treatment. The metabolic outcome glimpsed in normal sperm was an increased metabolic rate, while 'varicocele' sperm economized energy. Taken together, our results reveal DHT and AR as new players in sperm endocrinology, indicating that varicocele sperm may have difficulty in switching to the capacitated status. A decreased AR expression and a consequent reduced responsiveness to DHT in sperm may represent molecular mechanisms involved in the pathophysiology of varicocele leading to male infertility. This study revealed new detrimental effects of varicocele on sperm at the molecular level.

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Measurement of pancreatic lipase activity in serum by a kinetic colorimetric assay using a new chromogenic substrate

Cited by 90 publications

References 17 publications

The Angiopoietin-like Proteins ANGPTL3 and ANGPTL4 Inhibit Lipoprotein Lipase Activity through Distinct Mechanisms

The Angiopoietin-like Proteins ANGPTL3 and ANGPTL4 Inhibit Lipoprotein Lipase Activity through Distinct Mechanisms

Lipoprotein lipase activity and interactions studied in human plasma by isothermal titration calorimetry

Human sperm anatomy and endocrinology in varicocele: role of androgen receptor

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