The effect of the main nutrients and insulin on lipoprotein lipase activity in adipose tissue.
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Adipose Tissue↗
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Heparin-independent release of lipoprotein lipase activity from perfused rat hearts.
Heparin-independent release of lipoprotein lipase activity from isolated perfused rat hearts was measured and related to the rapid turnover of the enzyme. Hearts consistently released lipoprotein lipase activity (2.1 +/- 0.2 U/g released per min) during 60 min of nonrecirculating perfusion without heparin. This rate of release did not significantly differ from that measured in heparin-perfused hearts after the first 10 min of perfusion (2.2 +/- 0.2 U/g released per min). The fractional release rate of lipoprotein lipase activity during nonheparin perfusion was 1.3% per min, which was higher than that calculated for alkaline phosphatase (0.002%) and creatine kinase (0.03%) activities. The lipase activity released was activated 4-fold by serum and inhibited 94 and 88% by 0.5 M NaCl and 3 mg/ml protamine sulfate, respectively. Lipoprotein lipase activity in the 1-min heparin-releasable (extracellular) and residual (intracellular) compartment remained stable during the last 40 min of nonheparin perfusion. During this period total heart, intracellular and extracellular enzyme t1/2 were calculated to be 52, 42 and 10 min, respectively. The results are consistent with the postulate that continuous release of lipoprotein lipase into the vascular compartment may be an important determinant of its rapid turnover in the heart, and possibly other tissues.
Animals↗
Synthesis and secretion of lipoprotein lipase in heparan sulfate-deficient Chinese hamster ovary cells.
Synthesis and secretion of lipoprotein lipase was studied in two mutants of Chinese hamster ovary (CHO) cells which, due to a lack of xylosyl transferase (pgsA-745) or galactosyl transferase (pgsB-761), respectively, were deficient in heparan sulfate and chondroitin sulfate. One of the mutants (pgsB-761) was two- to threefold more active in synthesis and secretion of catalytically active lipoprotein lipase than the other mutant, which was about as active as the wild-type (K1) cells. A similar relation was found when lipoprotein lipase was metabolically labelled with 35S-methionine and then immunoprecipitated. Heparin stimulated secretion from all three cell types to a similar extent (about twofold). Heparin-releasable binding of 125I-labelled lipoprotein lipase was lower to either of the mutant cells than to the wild-type cells. Binding to the wild-type cells was reduced by heparitinase, while the low binding to the mutants was not affected. By immunogold labelling of cryosections, lipoprotein lipase was detected on the plasma membranes and on the inside of secretory vesicles of both wild-type and mutant cells, suggesting that some carrier could be involved. Inhibition of vesicular transport by monensin caused accumulation of lipoprotein lipase in the cells. In wild-type cells the lipase was mainly on the inside of vesicular structures, while in the mutants the main part was associated with membranous bodies that formed within the vesicles during a chase period. These results suggest that if lipoprotein lipase needs a carrier during intracellular assembly and transport, this function can be fulfilled by some structure other than heparan sulfate.
Animals↗
Enhancement of the binding of triglyceride-rich lipoproteins to the very low density lipoprotein receptor by apolipoprotein E and lipoprotein lipase.
The low-density lipoprotein (LDL) receptor plays a crucial role in cholesterol metabolism. A related protein, designated the very low density lipoprotein (VLDL) receptor, that specifically binds apolipoprotein (apo) E has recently been characterized and shown to be expressed in heart, muscle and adipose tissue and the human monocyte-macrophage cell line THP-1. The VLDL receptor binds and internalizes VLDL and intermediate density lipoprotein from Watanabe heritable hyperlipidemic (WHHL) rabbits as well as beta-migrating VLDL from cholesterol-fed rabbits but not LDL from WHHL rabbits. Chinese hamster ovary (CHO) cells transfected with the rabbit VLDL receptor cDNA have now been shown to bind or internalize VLDL (d < 1.006 g/ml) isolated from fasted normolipidemic human subjects with lower affinity than WHHL-VLDL or rabbit beta-VLDL. However, binding and internalization were markedly enhanced when fasted human VLDL was preincubated with either recombinant human apoE (3/3) or lipoprotein lipase (LPL) in CHO cells overexpressing the rabbit or human VLDL receptor. CHO cells transfected with both the rabbit VLDL receptor cDNA and the human LPL cDNA effectively bound, internalized, and degraded fasted human VLDL without pretreatment. Treatment of heparinase reduced the effect of LPL-mediated binding at 4 degrees C, but the inhibitory effect was lower at 37 degrees C. Pseudomonas LPL also enhanced the binding of human fasted VLDL to the VLDL receptor at 37 degrees C in CHO cells overexpressing the human VLDL receptor. Taken together, LPL causes the enhancement of triglyceride-rich lipoproteins binding to the VLDL receptor via both the formation of bridge between lipoproteins and heparan sulfate proteoglycans and its lipolytic effect. Ligand blot analysis showed that the apparent molecular mass of the VLDL receptor is 118 kDa, which is smaller than that of the LDL receptor. These results indicate that the VLDL receptor recognizes both triglyceride-rich lipoproteins that are also relatively rich in apoE, as well as the remnants of triglyceride-rich lipoproteins after catabolism and the interaction with heparan sulfate proteoglycans by LPL. The VLDL receptor may thus function as a receptor for remnants of triglyceride-rich lipoproteins in extrahepatic tissues.
Apolipoproteins E↗
Lipoprotein lipase of cultured mesenchymal rat heart cells. III. Effect of glucocorticoids and insulin on enzyme formation.
Lipoprotein lipase activity was studied in mesenchymal cells isolated from rat hearts and cultured for up to 8 days. The enzyme activity increased markedly between day 3 and 5 while the subsequent increase was less pronounced. Addition of hydrocortisone to complete culture medium resulted in an increase in lipoprotein lipase activity at all stages of culture. Lipoprotein lipase activity did not increase after addition of insulin to the complete culture medium. In the presence of serum-poor medium between day 3 and 6, the increase in lipoprotein lipase activity was much lower than in the presence of complete culture medium. Addition of hydrocortisone and insulin to the serum-poor medium resulted in a significant rise in lipoprotein lipase activity while less consistent effects were obtained after addition of each hormone alone. Transfer of cells to serum-poor medium between day 6 and 7 of culture caused a fall in enzyme activity. Addition of hydrocortisone alone and with insulin restored enzyme activity to control values. No effect on lipoprotein lipase was seen with estradiol, growth hormone, or glucagon when added to serum-containing medium, or serum-poor medium. These results indicate that the lipoprotein lipase of heart is controlled by glucocorticoids and that this control might require the presence of insulin for optimal expression.
Animals↗
Purification and characterization of lipoprotein lipase from pig myocardium.
1. Lipoprotein lipase was purified from pig myocardium by a two-step purification procedure involving (a) the formation of an enzyme-substrate complex and (b) affinity chromatography on Sepharose which contained covalently linked heparin. The purified enzyme gave in sodium dodecyl sulphate-polyacrylamide-gel electrophoresis one main band with an apparent molecular weight of 73 000. The enzyme, which was purified 70 000-fold, had a specific activity of 860 mumol of unesterified fatty acid liberated/h per mg of protein. 2. The purified enzyme hydrolysed [14C]triolein emulsions in the absence of added cofactors but its activity was increased fivefold by adding normal human serum. Of the low-density lipoprotein apoproteins only apolipoprotein CII could be substituted for serum in activating the enzyme. This lipase had maximum activity at 0.05-0.15 M-NaCl. Heparin increased the activity of the purified enzyme twofold at low concentrations, but high concentrations inhibited. The triglyceride lipase of pig myocardium thus resembles lipoprotein lipase purified from adipose tissue and from plasma, but is clearly different from pig hepatic triglyceride lipase.
Animals↗
Interactions between fatty acids and lipoprotein lipase: specific binding and complex formation.
Lipoprotein lipase is the extrahepatic lipase responsible for the hydrolysis of triglycerides in chylomicrons and very low-density lipoproteins. Its enzymic activity and its location on the surface of endothelial cells are affected by the presence of fatty acids, implying that the protein possesses binding sites for that ligand. In this study, we examine the binding of fatty acids to LpL and describe factors that must be considered when the dissociation constant of the acceptor-ligand equilibrium is close to the critical micelle concentration of the fatty acid. The interaction of fatty acids with lipoprotein lipase (LpL) was studied by two methods. A new direct method, based on the LpL-induced increase in the apparent critical micelle concentration of the sodium soap of the fatty acid, indicates the presence of multiple high-affinity binding sites. In the second method, the specific binding of fatty acids to LpL was measured by quantitating the blue shift in the tryptophan fluorescence of LpL that occurs upon binding the ligand. Both methods suggest the existence of 4-6 fatty acid binding sites on LpL with a dissociation constant on the order of 10(-6)-10(-7) M. Further analysis of the blue shift indicates that at higher concentrations of fatty acid, large complexes are formed consisting of 260-310 molecules of fatty acid per LPL monomer. In contrast, no large complexes are formed with fatty acids that form crystals above their solubility limit.
Animals↗
Regulation of macrophage lipoprotein lipase secretion by the scavenger receptor.
The effects of ligand binding to the scavenger receptor on the secretion of lipoprotein lipase by murine macrophages were examined. Inflammatory macrophages exposed to acetylated low-density lipoprotein (AcLDL) exhibited a dose-dependent, 40-80% increase in lipoprotein lipase secretion. This stimulation appeared to be unrelated to intracellular cholesterol and triacylglycerol levels and to phagocytosis in general. Resident and inflammatory macrophages treated with maleylated bovine serum albumin (Mal-BSA) showed a 3-fold increase in lipoprotein lipase secretion in a dose-dependent and time-dependent fashion. In contrast, dextran sulfate, which is another ligand recognized by the scavenger receptor, caused a dose-dependent decrease in lipoprotein lipase secretion. Casein, a ligand recognized by the Mal-BSA receptor, did not affect lipoprotein lipase secretion nor the ability of Mal-BSA to stimulate the enzyme, while dextran sulfate abolished the stimulatory effects of Mal-BSA. Since ethylamine, an inhibitor of receptor-mediated endocytosis, attenuated the increase in lipoprotein lipase secretion induced by AcLDL and Mal-BSA, but did not affect the inhibition induced by dextran sulfate, it is suggested that receptor-mediated endocytosis of ligands via the scavenger receptor might play a key role in the stimulation of lipoprotein lipase secretion in macrophages. This study reveals another mechanism for regulation of macrophage lipoprotein lipase secretion.
Animals↗
Instability of endothelium-bound lipoprotein lipase activity in perfused rat hearts.
The enzymatic activity of endothelium-bound lipoprotein lipase was measured in rat hearts perfused for 1 h at 37 degrees C. Viability parameters such as beating rate, flow rate, aortic pressure, and oxygen consumption were all kept strictly constant during the entire perfusion time. The lipase activity was determined by input-output difference of the triacylglycerol content in the nonrecirculating heart perfusate which contained either an artificial triolein emulsion or rat lymph chylomicrons. With either substrate, the lipase activity decreased with time: approximately 2% of initial lipase activity was lost per minute. The presence of rat serum (10%) in heart perfusate enhanced the rate at which the lipase activity disappeared. Only a small portion of the lipoprotein lipase activity, which was lost from the perfused heart, was recovered in the outflow perfusate. Our data demonstrate that, under our experimental conditions, the enzymatic activity of rat heart lipoprotein lipase does not remain constant during heart perfusion. Caution should therefore be taken by users of heart perfusion technique, especially for those who need constant lipoprotein lipase activity. The results suggest that the first step in the catabolic fate of endothelium-bound cardiac lipoprotein lipase is the loss of its catalytic activity. Whether the inactivated enzyme is released from the endothelium or remains in situ is not yet known.
Animals↗
Avian adipose lipoprotein lipase: cDNA sequence and reciprocal regulation of mRNA levels in adipose and heart.
cDNA clones for chicken adipose lipoprotein lipase were isolated from an expression library in lambda gt11 by antibody screening and characterized by hybridization selection and nucleotide sequencing. Based on the cDNA sequence and on N-terminal sequence analysis of the purified enzyme, chicken adipose lipoprotein lipase is a mature protein of 465 amino acids with a signal peptide of 19 or 25 amino acids, depending on which of two methionine residues is used for translation initiation. The predicted amino-acid sequence was found to be 73-77% identical to the four known mammalian adipose lipoprotein lipase sequences, with conservation of position of cysteine residues and putative functional domains, and number of potential N-glycosylation sites. Chicken lipoprotein lipase differs from mammalian lipoprotein lipases with respect to the position of one N-glycosylation site and the presence of an additional 15-17 C-terminal amino acids. 32P-labeled cDNA clones hybridized to mRNA species of 3.7 and 4.0 kb in Northern blots of heart and adipose, but not of liver RNA. In chickens that were fasted for 48 h and then refed, lipoprotein lipase mRNA levels in adipose increased to a maximal level of 350% that of controls at 10 h, whereas heart lipoprotein lipase mRNA levels fell to 40% of controls at 14 h. Concomitantly, no changes in total RNA were observed. Thus, avian lipoprotein lipase is subject to reciprocal pretranslational regulation in adipose and heart.
Adipose Tissue↗
Tissue-specific changes in lipid composition and lipoprotein lipase activity during the development of the chick embryo.
Lipoprotein lipase was present at a high specific activity in adipose tissue and heart of the chick embryo at the 14th day of development. The enzyme was also present in skeletal muscle but was absent from brain and liver. Major increases in the activity of lipoprotein lipase in adipose tissue and heart occurred from day 12 of development, concomitant with the beginning of the period of lipid uptake from the yolk. These results suggest that lipoprotein lipase may be involved in the utilisation of yolk-derived lipid by the tissues of the embryo. Relatively high levels of docosahexaenoic acid (22:6(n--3)) were present in the triacylglycerol isolated from plasma, adipose tissue, heart and liver. The relative proportions of this fatty acid in the triacylglycerol of the different tissues may be explicable in terms of the substrate specificity of lipoprotein lipase.
Adipose Tissue↗
Paracellular leakage of lipoprotein lipase across the mammary epithelium of the goat.
The ability of lipoprotein lipase to move across the mammary epithelium by a paracellular route was investigated. Five goats were milked hourly to activate the paracellular pathway. Three goats responded to hourly milking with a fivefold increase in milk lipoprotein lipase activity as compared with nonresponding goats. Massage of the mammary gland was necessary in the two nonresponding goats too cause increased lipoprotein lipase activity in milk. Oxytocin treatment during hourly milking also increased enzyme activity in milk from a nonresponding goat. Activation of the paracellular pathway by hourly milking increased milk sodium and protein and decreased potassium and lactose concentrations. After a 12-h milking interval, lipoprotein lipase activity was distributed primarily in the serum (48%) and cream (40%) fractions and, to a lesser extent, in the casein (12%) fraction. Hourly milking increased enzyme activity distributed in the serum fraction (62%), whereas enzyme activity associated with the cream (32%) and casein (6%) fractions decreased. Possible mechanisms for the origin of lipoprotein lipase in milk are discussed.
Animals↗
Lipoprotein lipase and sphingomyelinase synergistically enhance the association of atherogenic lipoproteins with smooth muscle cells and extracellular matrix. A possible mechanism for low density lipoprotein and lipoprotein(a) retention and macrophage foam cell formation.
Prominent features of atheromata include smooth muscle cells, cholesteryl ester-loaded macrophage foam cells, extracellular matrix, extracellularly trapped and aggregated lipoproteins, and various enzymes including lipoprotein lipase (LpL) and sphingomyelinase (SMase). The interplay of these factors was investigated in cell culture. Incubation of bovine aortic smooth muscle cells for 18 h at 37 degrees C with low density lipoprotein (LDL) in the presence of LpL and SMase led to massive aggregation of LDL on the surface of the cells as viewed by phase, fluorescence (using 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate-LDL), and electron microscopy. This aggregation required both enzymes. Studies with 125I-LDL confirmed these observations: 125I-LDL cell association in the presence of LpL plus SMase was 50-100-fold greater than in the absence of the two enzymes and was 10-fold greater than in the presence of either enzyme alone. A similar effect (68-fold enhancement) was seen with 125I-labeled lipoprotein(a) (Lp(a)), another atherogenic lipoprotein. In all cases, 125I-lipoprotein degradation was relatively low (< 5% of cell-associated material). LpL/SMase-mediated association of 125I-LDL with smooth muscle cells was still observed when enzymatically inactive LpL was used. The effect was markedly diminished when the smooth muscle cells were treated with a combination of chondroitin ABC lyase and heparitinase or when mutant Chinese hamster ovary cells that lack cell-surface proteoglycans were used, indicating a specific role for cellular proteoglycans. When smooth muscle cells with 125I-LDL or 125I-Lp(a) aggregates were rinsed and then coincubated with mouse peritoneal macrophages for a further 24 h, visible aggregates disappeared, and there was marked 125I-lipoprotein degradation. Electron micrographs after 24 h of co-culture showed lipid-laden, foamy macrophages situated on top of smooth muscle cells, suggesting that the macrophages phagocytosed and metabolized the smooth muscle cell-associated LDL aggregates. Last, 125I-LDL association with smooth muscle cell extracellular matrix was also synergistically enhanced by LpL and SMase, to a level that was 19-fold greater than in the absence of the two enzymes. Thus, the interaction of LDL and Lp(a) with four atheroma components, namely, smooth muscle cells, extracellular matrix, LpL, and SMase, represents a physiologically plausible mechanism for massive, focal retention and aggregation of atherogenic lipoproteins in the arterial wall with subsequent macrophage foam cell formation.
Animals↗
Is decreased activity of C-II activated lipoprotein lipase in type III hyperlipoproteinemia (broad-beta-disease) a cause or an effect of increased apolipoprotein E levels?
Apolipoprotein E (ApoE; "arginine-rich" polypeptide) strongly inhibited both C-I and C-II activated lipoprotein lipases but not the protamine insensitive triglyceride lipase. Inhibition of lipoprotein lipases by ApoE in contrast to inhibition by C-III was not reversed to any significant extent by either increased concentration of activator or triglyceride in the substrate. Our previous studies have shown that in a type III hyperlipoproteinemia (broad-beta-disease) a post-heparin plasma lipoprotein lipase activated by C-II polypeptide of lipoprotein C is decreased in enzyme activity and exhibits an impaired ability to hydrolyze triglycerides in very low density lipoproteins. Type III patients are characterized by elevated concentrations of ApoE in the serum. The data presented in this report suggest that the decreased C-II activated lipoprotein lipase may be further aggravated by increased ApoE levels. Since this enzyme is involved in the catabolism and removal of lipoproteins, decreased activity of C-II activativated lipoprotein lipase may presumably be responsible for increased ApoE.
Apolipoproteins↗
Comparison of lipoprotein lipase activity in heart myocytes and perfused hearts.
This study was initiated to compare lipoprotein lipase activity in isolated heart myocytes and the heparin residual compartment of perfused hearts from adult rats. Heart lipoprotein lipase activity was divided into two fractions by 1 min of heparin perfusion. Heparin-residual and myocyte lipoprotein lipase activity were lower in hearts obtained from fasted compared to fed rats. In each case, the myocyte enzyme activity was 55 to 60% of heparin-residual levels. The difference between myocyte and heparin-residual activity may be a consequence of the time and treatment required to isolate cells in that long-term in vitro exposure of heart tissue to heparin also reduces residual activity. In vivo treatment with endotoxin decreased both heparin-residual and myocyte lipoprotein lipase activities; whereas, colchicine administration increased both activities compared to saline injected rats. In this latter experiment heart heparin-residual and myocyte lipoprotein lipase activities were positively correlated (r = 0.90). The results indicate that in the mature heart intracellular lipoprotein lipase activity is primarily associated with myocytes.
Animals↗
Apolipoprotein E and lipoprotein lipase secreted from human monocyte-derived macrophages modulate very low density lipoprotein uptake.
We investigated the roles of lipoprotein lipase and apolipoprotein E (apoE) secreted from human monocyte-derived macrophages in the uptake of very low density lipoproteins (VLDL). ApoCII-deficient VLDL were isolated from a patient with apoCII deficiency. The lipolytic conversion to higher density and the degradation of the apoCII-deficient VLDL by macrophages were very slight, whereas the addition of apoCII enhanced both their conversion and degradation. This suggests that the lipolysis and subsequent conversion of VLDL to lipoproteins of higher density are essential for the VLDL uptake by macrophages. VLDL incubated with macrophages obtained from subjects with E3/3 phenotype (E3/3-macrophages) showed a 17-fold greater affinity in inhibiting the binding of 2 micrograms/ml 125I-low density lipoprotein (LDL) to fibroblasts than native VLDL, whereas the incubation of VLDL with macrophages obtained from a subject with E2/2 phenotype (E2/2-macrophages) did not cause any increase in their affinity. Furthermore, 3 micrograms/ml 125I-VLDL obtained from a subject with E3/3 phenotype were degraded by E3/3-macrophages to a greater extent than by E2/2-macrophages (2-fold), indicating that VLDL uptake is influenced by the phenotype of apoE secreted by macrophages. From these results, we conclude that both lipolysis by lipoprotein lipase and incorporation of apoE secreted from macrophages alter the affinity of VLDL for the LDL receptors on the cells, resulting in facilitation of their receptor-mediated endocytosis.
Apolipoprotein C-II↗
Enhanced release and synthesis of lipoprotein lipase in rat heart cell cultures exposed to high concentrations of Hepes.
While attempting to optimize conditions for synthesis of lipoprotein lipase by cultured heart cells, we encountered an unexpected rise in enzyme activity when media were supplemented inadvertently with 100 mM Hepes buffer (4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid). This finding was further investigated and optimal results were obtained at pH 7.0-7.2. The increase in lipoprotein lipase activity was time dependent; after 3-6 h there was a rise in medium activity but cellular activity increased only after 24 h. The increased enzyme activity was defined as lipoprotein lipase by inhibition with antiserum to rat adipose tissue lipoprotein lipase. A 72-h exposure to Hepes resulted in a 30% increase in the incorporation of [35S]methionine into cellular proteins and a 2-fold increase into heparin-releasable proteins. Using heparin Sepharose chromatography and stepwise elution, a lipoprotein lipase enriched fraction was recovered with 2 M NaCl. The amount of [35S]methionine and [3H]galactose incorporated into protein of this fraction derived from Hepes-treated cells was 2-6-fold that of controls. A 4-fold increase in cellular lipoprotein lipase mass in Hepes-treated cells was shown by immunoblotting. Results obtained with Hepes-conditioned medium suggest the presence of cell-derived compounds that enhance release and subsequent synthesis of lipoprotein lipase. The effect of Hepes-conditioned medium on lipoprotein lipase resembled to some extent that of the addition of heparin. Therefore, it appears that when Hepes is first added to the culture medium, it might promote a release of heparan sulfate or related compounds, possibly by virtue of its negatively charged sulfonic acid residue. The accumulated heparan sulfate could then promote a sustained release of lipoprotein lipase into the culture medium which in turn leads to increased enzyme synthesis.
Animals↗
The relationship between the basal lipolytic and lipoprotein lipase activities in human adipose tissue.
The basal rate of lipolysis and basal lipoprotein lipase activity were determined in vitro in subcutaneous adipose tissue obtained from eight healthy non-obese subjects, ten obese subjects before and during one week's starvation, nine untreated non-insulin dependent diabetics and seven treated non-insulin dependent diabetics whose disease had been under metabolic control for at least three months. There was a negative correlation between the rate of lipolysis and activity of lipoprotein lipase in untreated diabetes mellitus and during starvation (r from -0.87 to -0.81). Under these two conditions the rate of lipolysis is increased and the lipoprotein lipase activity is decreased. There was no correlation between lipolysis and lipoprotein lipase in non-obese subjects, non-starving obese subjects and treated diabetic patients (r from 0.11 to 0.36). Thus, during starvation and in untreated diabetes, there is a strong reciprocal relationship between basal lipolytic activity and basal lipoprotein lipase activity in human adipose tissue which is not found under normal conditions or in obesity and well-controlled diabetes. It is concluded that a negative connection between lipolysis and lipoprotein lipase in human adipose tissue may be of physiological importance for the regulation of the energy balance in conditions such as untreated non-insulin dependent diabetes and starvation where adipose tissue lipids are the major source of energy.
Adipose Tissue↗