Lipase Complex Research Articles

Impaired adipocyte lipolysis in nonobese women with the polycystic ovary syndrome: a possible link to insulin resistance?

The polycystic ovary syndrome (PCOS) is the most common hyperandrogenic disorder among women and is characterized by metabolic and cardiovascular aberrations similar to those seen in the so-called insulin resistance syndrome. The regulation of lipolysis was investigated in isolated abdominal sc adipocytes from 10 nonobese women with PCOS and in 11 age- and body mass index-matched healthy women. Eight PCOS women were reinvestigated after 3 months of treatment with combined oral contraceptives containing ethinyl estradiol and norethisterone, which normalized hyperandrogenicity. The PCOS women showed a marked resistance to the lipolytic effect of noradrenaline due to defects at two different levels in the lipolytic cascade: first, a 7-fold reduction in sensitivity to the beta 2-selective agonist terbutaline (P < 0.005), which could be ascribed to a 50% lower beta 2-adrenoceptor density (P < 0.02) as determined with radioligand binding; there was no difference with regard to dobutamine (beta 1) or clonidine (alpha 2-sensitivity) or beta 1-adrenoceptor density; second, the maximum lipolytic response was also 35% lower (P < 0.02) in the PCOS women compared to that in the healthy women. This was seen with all beta-adrenergic agonists and the postreceptor-acting agents forskolin (activating adenylyl cyclase) and dibutyryl cAMP (activating protein kinase). Neither beta 2-adrenoceptor sensitivity or density nor the reduced lipolytic responsiveness was restored by 3 months of oral contraceptives treatment. The results indicate the existence of a marked impairment of catecholamine-induced lipolysis in nonobese PCOS women displaying early features of the insulin resistance syndrome due to multiple lipolysis defects as a lower beta 2-adrenoceptor density and reduced function of the protein kinase, hormone-sensitive lipase complex. These lipolysis defects are identical to those observed in the insulin resistance (metabolic) syndrome and could be a primary pathogenic mechanism for the development of these disorders.

Surfactant‐Modified lipase for the catalysis of the interesterification of triglycerides and fatty acids

The lipase-catalyzed intresterification of triglycerides and fatty acids in n-hexane was studied. Initially, lipase Saiken was modified with a surfactant of sorbitan esters so that its dispersibility in hydrophobic organic media was improved. The surfactant-modified lipase formed in the modification process carried out in a buffer solution has 1,3-positional specificity and predominantly catalyzed the interesterification reaction in a microaqueous n-hexane system. The modification technique converted inactive lipases to very active biocatalysts for the interesterification of triglycerides and fatty acids. The pH and the weight ratio of surfactant to enzyme used during the lipase modification process have shown significant effects in determining the recoveries of the protein and enzyme activity from the buffer solution, the protein content of the modified lipase complex after being freeze dried, and the interesterification activity of the complex. The water content in the reaction solution has strongly influenced the enzyme activity as well as the distribution of the products. (c) 1995 John Wiley & Sons, Inc.

Association with natural polymers modifies lipase selectivity

Abstract It was shown in the previous communication that the specificity of lipases can be altered through physico‐chemical modifications with defined polymers. The same effect is achieved when lipases form associations with natural polymers. In this communication, non‐covalent modifications of lipases with other polymers, such as carbohydrates and proteins, were investigated and again a correlation between the polarity of the polymer and that of the liberated fatty acids was established. This was most clearly seen following hydrolysis of the pectin in a pectin‐lipase associate: The ratio of liberated oleic‐ to stearic acid changed from 2.2:1 to 1:1 when pectin hydrolysis was stopped. The natural oleic versus stearic ratio of the pure lipase was 0.5:1. Use of a model system consisting of triglycerides containing only one fatty acid provided an explanation for modified lipase specificity. It is suggested that the potential penetration depth of the lipase complex does not determine the profile of liberated ...

Adrenergic regulation of lipolysis in human fat cells during exercise

The adrenergic regulation of lipolysis was studied, before and after 30 min of submaximal exercise, in isolated adipocytes removed from the abdominal and gluteal regions of healthy non-obese men and women. Noradrenaline-induced lipolysis was significantly enhanced in gluteal adipocytes from men but not in women after exercise. However, the pure beta-adrenergic responsiveness was equally increased in gluteal adipocytes of both sexes after exercise, as judged by the effect of isoprenaline. Furthermore, the alpha 2-adrenergic anti-lipolytic responsiveness was more apparent after exercise in females than in males thereby counter-balancing the increase in the beta-adrenergic effect in the gluteal region in the former. The increased beta-adrenergic responsiveness induced by exercise in gluteal adipocytes of males could be mimicked by agents acting at the levels of adenylate cyclase, coupling proteins, phosphodiesterase, and protein kinase and seems to be due to an adaptive enhancement at the hormone-sensitive-lipase level. There was no change in the stoichiometric properties of beta-adrenoceptors of gluteal adipocytes after exercise. Abdominal adipocytes of both sexes were four to five times more responsive to noradrenaline than gluteal ones. However, exercise induced no further enhancement of the catecholamine-stimulated lipolysis rate in fat cells from this site. Thus, the influence of exercise on catecholamine-stimulated lipolysis is regional and sex dependent. Men, but not women, have a greater ability to adapt lipolysis to increasing energy demands during exercise that are due to an acute increase in the effectiveness of the hormone-sensitive lipase complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Catecholamine-induced lipolysis in adipose tissue of the elderly.

Age-dependent alterations in the effects of catecholamines on lipolysis were investigated in 25 young (21-35 yr) and 10 elderly (58-72 yr) healthy, nonobese subjects using isolated adipocytes obtained from abdominal subcutaneous tissue. Basal lipolysis was not affected by aging, while the rate of catecholamine-stimulated lipolysis was reduced by 50% in the elderly subjects (P less than 0.005). To elucidate the mechanisms behind this phenomenon lipolysis was stimulated with agents that act at well-defined steps in the lipolytic cascade, from the receptor down to the final step: the activation of the protein kinase/hormone-sensitive lipase complex. All agents stimulated lipolysis at a 50% lower rate in elderly as compared with young subjects (P less than 0.05 or less). However, half-maximum effective concentrations of the lipolytic agents were similar in both groups. The antilipolytic effects of alpha 2-adrenoceptor agonists were also the same in young and old subjects. Moreover, the stoichiometric properties of the beta- and alpha 2-receptors did not change with increasing age. In vivo studies performed on the same individuals likewise demonstrated an impaired lipolytic responsiveness, with 50% lower plasma glycerol concentrations during exercise in the elderly subjects (P less than 0.05), in spite of a normal rise in plasma norepinephrine. The plasma glycerol levels correlated strongly to the glycerol release caused by catecholamine-stimulated lipolysis in vitro in both young and elderly subjects (r = 0.8-0.9, P less than 0.001). In conclusion, a decreased activation of the hormone-sensitive lipase complex appears to be the mechanism underlying a blunted lipolytic response of fat cells to catecholamine stimulation in elderly subjects. This finding may, explain the age-dependent decreased lipolytic response to exercise in vivo.

Large agarose-lipase beads for the hydrolysis of triglycerides

Pancreatic lipase could be immobilized by binding to large agarose beads or by entrapment within a polyacrylamide polymer. Large agarose beads, prepared from native agarose, were preferred for their higher binding capacity and easy intact separation for reuse. The agarose lipase complex is stable for 6 months at 4 °C and pH 7.5. The immobilized lipase preparation maintained 80% of its initial activity even after reusing it eight times. Consequently, the immobilized lipase was recommended for semicontinuous hydrolysis of olive oil and milk fat trigylcerides. The amount of total hydrolysis was 31 ± 1.17% for milk fat compared with 27 ± 1.03 % for olive oil.

Large agarose-lipase beads for the hydrolysis of triglycerides

Candida cylindracea lipase could be immobilized by binding to large agarose beads or by entrapment within polyacrylamide polymer. Large agarose beads, prepared from native agarose, were preferred for their higher binding capacity and easy intact separation for reuse. The agarose-lipase complex is stable for 6 months at 4°C, maintaining 80% of its initial activity despite eight-fold re-use. Consequently, the lipase complex is recommended for semi-continuous hydrolysis of olive oil and milk fat triglycerides. The extent of total hydrolysis was 30·8 ± 1·25% for olive oil as against 23·1 ± 0·95% for milk fat.

Conformational change in pancreatic lipase induced by colipase

Pancreatic lipase is a key enzyme for the intraluminar digestion of fats. The adsorption of lipase to the substrate which occurs readily when the particles are composed of pure triacylglycerol is prevented by the accumulation at the interface of various amphiphiles like bile salt and phospholipids present in the duodenum during fat digestion. In the presence of pancreatic colipase the activity of lipase is, however, fully restored. The exact mechanism for this reactivation is not known. Two different mechanisms have been suggested. The first considers colipase as an ‘anchor’ for lipase at the hydrophilic interface. In a series of sequential steps colipase will alone recognize and bind to the interface and from there bind to lipase thus anchoring the enzyme back to its triacylglycerol substrate [ 1,2]. The other claims that none of the proteins could bind to the interface but that lipase and colipase form a complex with bile-mixed micelles in solution. This complex is then adsorbed as a single unit by virtue of the bile lipids to the triacylglycerate interface 131. Lipase is known to form a 1: 1 molar complex with colipase [4]. The binding which is both electrostatic and hydrophobic has a dissociation constant of 5 x lop7 M in buffer [5]. In bile salt solution the binding is weaker (&l = 3 X 10 -6 M). However, in the presence of mixed oleic acidbile salt micelles the binding is strongly increased [6] with a Kd of lo* M [7]. In the lipase/colipase binding there has been no indication for any conformational change when the interaction has been studied in buffer or in bile salt solution as indicated by CD measurements 14). This may, however, be due to weak binding between the two proteins under these conditions which makes necessary the use of concentrated solutions to assure a binding. Recently, a covalent complex of lipase and colipase was obtained by use of a crosslinking agent [8]. This complex had binding properties to a mixed phospholipid-triacylglycerol interface that could not be explained as an effect of colipase alone but suggested that’colipase had somehow induced a conformational change in lipase enabling lipase to take part in the substrate binding. This study shows that colipase can induce a conformational change in lipase seen as an ultraviolet spectral shift of the complex compared to the single proteins. The change was -5-fold greater in the presence of mixed oleic acid-bile salt micelles. At pH 5, the addition of bile salt to lipase alone also induced a spectral shift. The mechanism for colipase reactivation of lipase is discussed in the light of the above experiments.

Effect of dextran sulfate on the interaction between very low density lipoprotein and lipoprotein lipase

The effect of dextran sulfate on the interaction between very low density lipoprotein (VLDL) and purified bovine milk lipoprotein was studied. Dextran sulfate increased VLDL-triacylglycerol hydrolysis by lipoprotein lipase about 2-fold, but did not alter the K m value for triacylglycerol in VLDL. Strong association of dextran sulfate with the VLDL-lipoprotein lipase complex was demonstrated by gel filtration on BioGel A-5m, although dextran sulfate did not bind to VLDL and only very slightly to lipoprotein lipase. These findings suggest that dextran sulfate increases triacylglycerol hydrolysis in VLDL by binding to the VLDL-lipoprotein lipase complex.

Dissociation of the lipid-enzyme complex of hormone-sensitive lipase using high density lipoprotein or apolipoprotein A-I

Hormone-sensitive lipase in homogenates of adipose tissue occurs as a large, lipid-rich complex including several acylhydrolase activities that emerge quantitatively in the void volume on gel filtration chromatography (2% agarose). Incubation with intact human plasma high density lipoprotein or with lipidfree apolipoprotein A-I, however, disrupted the lipid-rich complex almost completely and most of the enzyme activity eluted from a 2% agarose column at about Ve = 2.3 × Vo. This use of the detergent-like properties of apolipo-protein A-I may be of value for dissociation of other lipid-associated or membrane-bound enzymes.

Possible roles of bile lipids and colipase in lipase adsorption.

The adsorption isotherms of bile salts, phospholipids, and cholesterol were determined with siliconized glass beads. It was observed that the molar fractions of cholesterol, phospholipid, and bile polypeptide fractions increased simultaneously and considerably on the surface of the beads in comparison to the corresponding fractions found in bile. The composition of the adsorbed film is approximately 1 cholesterol: 2 phospholipid: 3 bile salt molecules. The performed complex of lipase, colipase, and bile lipids behaves as an entity which determines lipase adsorption. The modification of the interface quality of a lipid substrate by a detergent is not perse the reason for the lack of lipase adsorption. A model is proposed according to which lipolysis under physiological conditions would occur in two steps requiring two cofactors. Colipase would be necessary for the formation of the lipase-bile lipoprotein complex, and bile lipids would be required to direct the adsorption of this lipolytic entity toward the emulsified substrate.

Activatable cholesterol esterase and triacylglycerol lipase activities of rat adrenal and their relationship

Activatable cholesterol esterase and triacylglycerol lipase of rat adrenal were 58–69% recovered in the 100 000 × g supernatant fraction. Activatable triacylglycerol lipase activity was differentiated from the activity of acid lipase and lipoprotein lipase also found in this fraction. Cholesterol esterase was activated 39.7 ± 13.6% (S.D.) and triacylglycerol liapse 11.9 ± 2.9% in a reaction dependent on ATP, cyclic AMP, and protein kinase. The two activities were shown by differential inhibition by an organophosphate, and by partial separation on salting out, to be largely due to separate enzymes. The two enzymes bound tightly to substrate emulsions with quantitatively similar distribution between competing emulsions, suggesting concerted binding. Coinciding gel filtration patterns reinforced, the hypothesis of a lipase complex. Cholesterol esterase comprised a major component of higher apparent K m for substrate and molecular weight 3 · 10 5–6 · 10 5 by gel filtration, and a minor component of lower apparent K m and heterogeneous molecular weight above 1 million, which was found mostly in complex with lipid.

Enzyme-Substrate Complex of Pancreatic Lipase and Its Artificial Substrates, Riboflavin Di- and Tributyrate

Enzyme-Substrate Complex of Pancreatic Lipase and Its Artificial Substrates, Riboflavin Di- and Tributyrate Get access Kunio YAGI, Kunio YAGI Institute of Biochemistry, Faculty of Medicine, University of NagoyaNagoya Search for other works by this author on: Oxford Academic PubMed Google Scholar Mayumi OSAMURA, Mayumi OSAMURA Institute of Biochemistry, Faculty of Medicine, University of NagoyaNagoya Search for other works by this author on: Oxford Academic PubMed Google Scholar Nobuko OHISHI Nobuko OHISHI Institute of Biochemistry, Faculty of Medicine, University of NagoyaNagoya Search for other works by this author on: Oxford Academic PubMed Google Scholar The Journal of Biochemistry, Volume 73, Issue 2, February 1973, Pages 455–457, https://doi.org/10.1093/oxfordjournals.jbchem.a130101 Published: 01 February 1973 Article history Received: 11 August 1972 Published: 01 February 1973