Secrete Lipoprotein Lipase Research Articles

Angptl4 links α-cell proliferation following glucagon receptor inhibition with adipose tissue triglyceride metabolism

Type 2 diabetes is characterized by a reduction in insulin function and an increase in glucagon activity that together result in hyperglycemia. Glucagon receptor antagonists have been developed as drugs for diabetes; however, they often increase glucagon plasma levels and induce the proliferation of glucagon-secreting α-cells. We find that the secreted protein Angiopoietin-like 4 (Angptl4) is up-regulated via Pparγ activation in white adipose tissue and plasma following an acute treatment with a glucagon receptor antagonist. Induction of adipose angptl4 and Angptl4 supplementation promote α-cell proliferation specifically. Finally, glucagon receptor antagonist improves glycemia in diet-induced obese angptl4 knockout mice without increasing glucagon levels or α-cell proliferation, underscoring the importance of this protein. Overall, we demonstrate that triglyceride metabolism in adipose tissue regulates α-cells in the endocrine pancreas.

Oxidized type IV hypertriglyceridemic VLDL-remnants cause greater macrophage cholesteryl ester accumulation than oxidized LDL

We have previously shown that very low density lipoproteins (VLDL, Sf 60-400) from subjects with type IV hyperlipoproteinemia (HTG-VLDL) will induce appreciable cholesteryl ester accumulation in cultured macrophages (J774A.1). The present study examined whether copper-mediated oxidative modification of HTG-VLDL and their remnants would further enhance cholesteryl ester accumulation in J774A.1 cells. Incubation with oxidized VLDL-remnants caused the greatest increase in cellular cholesteryl ester concentrations (54-fold) relative to control cells (P = 0.001). HTG-VLDL and VLDL-remnants each induced similar increases in cholesteryl ester levels (32.3- and 35.8-fold, respectively; both P = 0.001), whereas incubation with oxidized HTG-VLDL brought about only a 20.6-fold increase in cholesteryl ester concentrations (P = 0.014). The increase in cellular cholesteryl ester concentrations induced by oxidized VLDL-remnants was significantly higher (P < or = 0.04) than that induced by all other lipoproteins tested including low density lipoprotein (LDL) and oxidized LDL which caused a 6.7- and a 35.1-fold increase (P < or = 0.0002 for both), respectively. Unlike HTG-VLDL and to a lesser extent VLDL-remnants, uptake of oxidized VLDL and oxidized VLDL-remnants did not require catalytically active, cell secreted lipoprotein lipase. Co-incubation with polyinosine, which blocks binding to the type I scavenger receptor, completely inhibited the cholesteryl ester accumulation induced by oxidized HTG-VLDL, oxidized VLDL-remnants and oxidized LDL (P < or = 0.02). We conclude that oxidation of VLDL-remnants significantly enhances macrophage cholesteryl ester accumulation compared to either HTG-VLDL, VLDL-remnants, or oxidized LDL. Uptake of oxidized VLDL and oxidized VLDL-remnants does not require catalytically active lipoprotein lipase, and involves a receptor that can be competed for by polyinosine.

Cellular and secreted lipoprotein lipase revisited

Lipoprotein lipase (LPL) of adipose cells is present only in membrane compartments, mainly in the Golgi apparatus. LPL is a typical secretory protein which appears to be active as a homodimer. The process of LPL synthesis and maturation requires multiple steps. LPL is synthesized in the endoplasmic reticulum as an inactive monomer of Mr 51,000; a high-mannose, inactive monomer of Mr 55,500 is then formed. An active homodimer form, bearing two complex oligosaccharide chains per monomer of Mr 58,000, forms in the Golgi apparatus. This mature form, present in secretory vesicles, can be secreted constitutively or after exposure to heparin. A model is proposed in which LPL is present in secretory vesicles in a potentially active, condensed, or "polymerized" form. This model, which applies to various LPL-containing tissues in different species - including human - would explain the "activation" of LPL.

Dependence on Ca2+ of Lipoprotein Lipase Stability

Macrophages continuously secrete lipoprotein lipase (LPL) into the culture medium. When LPL was collected from thioglycollate-elicited peritoneal macrophages (Tg-Mø) or J774.1 cells over a 4 h period in Ca2+ and Mg2(+)-free Dulbecco's modified Eagle's medium (d-DMEM) the activity in the collection medium was reduced by 40-62% and 23%, respectively, as compared to that expressed in full medium (DMEM). Ca2+ supplementation during the collection period in d-DMEM augmented LPL activity in the medium; about 1 mM Ca2+ was required for attainment of activity comparable to that expressed in DMEM. Addition of Ca2+ during the assay did not enhance LPL activity collected into d-DMEM. Addition of EGTA to the assay mixture reduced LPL activity by 34-60% and when present in the collection medium, EGTA led to a reduction in enzyme activity greater than 90%. A 4 h incubation of Tg-Mø in 3 mM EGTA led to an almost complete loss of intracellular Ca2+ (measured by efflux of 45Ca2+ from preloaded cells), yet there was no change in the overall synthesis and secretion of proteins and in the phagocytic capability of the cells. LPL activity in the enzyme collection medium after its removal from cell monolayers was stable at least up to 4 h at 0 degrees C and at 23 degrees C. Activity was progressively lost with increased temperatures: up to 40% loss at 37 degrees C in 4 h. Addition of EGTA to the above medium led to an enhanced rate of irreversible enzyme inactivation: 76-86% loss of activity in 4 h at 37 degrees C. No inactivation was observed at 0 degrees C and at 23 degrees C in the presence of EGTA. The results indicate a critical role for Ca2+ in enzyme stabilization.

Effects of cytokines on the production of lipoprotein lipase in cultured human macrophages.

Macrophages are important cells in the pathogenesis of atherosclerosis because of their tendency to accumulate lipid and become transformed into foam cells. Cultured human monocyte-derived macrophages spontaneously secrete lipoprotein lipase (LPL), and LPL has been linked to increased lipid uptake by these cells. Because secretion of various macrophage products depends on activation by lymphokines, we studied the effects of immunoregulatory lymphokines on LPL secretion by cultured human macrophages. After culturing cells in RPMI 1640 medium with 20% fetal calf serum, recombinant human gamma-interferon (gamma-INF), interleukin-1 (IL-1), and interleukin-2 (IL-2) were added to the medium and LPL secretion was assessed by measuring LPL activity and/or LPL mass in the medium. Gamma-INF suppressed LPL production both when added to freshly plated cultures of human blood monocytes, as well as when added to monocyte/macrophages from mature cultures (day 6) that were producing large amounts of LPL. IL-1 inhibited medium LPL when added to freshly plated cultures, but not when added to mature cultures. On the other hand, IL-2 did not inhibit LPL in freshly plated cultures, but produced a dose-dependent suppression of LPL from mature cultures. None of the cytokines were cytotoxic to macrophages, and cells that were cultured in gamma-INF demonstrated partial recovery from LPL-suppressive doses of the cytokine. After exposure of cells to 50 U/ml of gamma-INF and 50 U/ml of IL-2 for 3 days, LPL mRNA levels, when expressed as LPL/gamma-actin ratios, were 42% and 53% of controls, respectively. Thus, activation of human macrophages in vitro by gamma-INF resulted in a suppression of LPL production.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of lipoprotein lipase activity in mouse peritoneal macrophages by recombinant human tumor necrosis factor

Thioglycollate-elicated mouse peritoneal macrophages spontaneously secrete lipoprotein lipase during culture. Exposure of the cultures to 50 ng/ml of recombinant human tumor necrosis factor (rTNF) for 48 h resulted in a 69% reduction in lipoprotein lipase activity in the culture medium with a concomitant decrease in cellular enzyme activity. The decrease in enzyme activity was not the result of rTNF-dependent reduction in the total protein synthesis, since the presence of rTNF did not affect [ 3H]leucine incorporation into cellular proteins. The effect of rTNF on lipoprotein lipase was reversible; upon TNF withdrawal, enzyme activity returned to basal levels after 60 h. The reduction of lipoprotein lipase in rTNF-treated cultures could be completely prevented by preincubation with a specific antiserum against recombinant human TNF. The late onset of decrease of lipoprotein lipase (LPL) activity suggests that rTNF might induce a mediator, which in turn suppresses LPL production. While rTNF was very effective in reducing lipoprotein lipase activity in mouse peritoneal macrophages, it did not affect lipoprotein lipase activity when added to the murine J774 cell line and to CT 2 macrophage-like cells, a variant of the J774 cell line.

Bacterial lipopolysaccharide reduces macrophage lipoprotein lipase levels: an effect that is independent of tumor necrosis factor

Human monocyte-derived macrophages secrete lipoprotein lipase (LPL) in culture. The regulation of human macrophage LPL production is poorly understood. Since bacterial lipopolysaccharide (LPS) alters production of several macrophage secretory products, its effect on human monocyte-derived macrophage LPL was tested. LPS treatment produced a dramatic dose-dependent decrease in LPL activity in macrophage-conditioned media. At 100 ng/ml LPS, medium LPL activity dropped by 60%. The effect of LPS on macrophage LPL activity was rapid, was blocked by polymixin B, and was not due to cytotoxicity. LPS lowers (by about 60%) the steady state level of LPL mRNA, suggesting that its effect is exerted at the level of mRNA metabolism. Since LPS stimulates macrophage production of cachectin/tumor necrosis factor (TNF), a potent inhibitor of LPL production by the 3T3-L1 adipocyte-like cell line, it was determined whether TNF reduces macrophage LPL levels. Treatment of human macrophages with up to 1000 U/ml of recombinant human TNF had no effect on macrophage LPL activity. When TNF was added in combination with LPS, no additional effect on LPL activity was observed over that seen with LPS alone. Furthermore, the LPS effect was not blocked by a monoclonal anti-TNF antibody. Thus, bacterial LPS potently decreases macrophage LPL activity and mass independent of an autocrine effect of TNF.

High-cholesterol diet-induced lipoproteins stimulate lipoprotein lipase secretion in cultured rat alveolar macrophages

We have previously shown that cultured rat alveolar macrophages synthesize and secrete lipoprotein lipase into the medium. The purpose of the present experiments is to examine whether cholesterol-enriched lipoproteins from cholesterol-fed animals have any effects on the lipoprotein lipase secretion and the lipid accumulation in macrophages. Macrophages incubated with the VLDL obtained from rats fed a normal diet secreted 2-fold higher amounts of lipoprotein lipase than those without lipoproteins. Intermediate-, low- and very-low-density lipoproteins from rats fed a high-cholesterol diet also enhanced the lipoprotein lipase secretion. Normal high- and low-density lipoprotiens, and high-density lipoproteins from hypercholesterolemic animals did not cause any increase in the lipoprotein lipase secretion. The lipoproteins which stimulated the lipoprotein lipase secretion caused intracellular accumulation of both triacylglycerol and cholesterol. It is speculated that macrophages residing in the environment rich in lipoproteins, especially hypercholesterolemic lipoproteins, take them up and accumulate lipids intracellularly, and that this process links with the lipoprotein lipase secretion. The secreted lipoprotein lipase could facilitate, by degrading lipoproteins, the uptake of lipoprotein lipase-modified lipoproteins. Probably such a series of events is of importance in the foam cell formation of macrophages.

Regulation of lipoprotein lipase secretion by mouse peritoneal macrophages

Resident mouse peritoneal macrophages in culture spontaneously secrete lipoprotein lipase. Secretion of lipoprotein lipase was 10-fold greater in thioglycollate-elicited and 6-fold greater in mineral oil-elicited macrophages. The increase in enzyme secretion was dependent on protein synthesis and glycosylation of the newly synthesized enzyme. The addition of 1 to 500 ng per ml of lipopolysaccharide to the culture medium resulted in a dose-dependent inhibition of lipoprotein lipase secretion. Thus, secretion of macrophage lipoprotein lipase is a regulated process, and may be increased as part of the response to inflammation. This may have important implications in the atherogenic process in the arterial wall.

Effect of lipoprotein on LPL activity of guinea pig alveolar macrophages

Macrophages may play an important role in the atherogenic process because they appear to be precursors of some arterial wall foam cells. Recently, it has been reported that human peripheral monocytes in culture synthesize and secrete lipoprotein lipase (LPL). LPL catalyzes the hydrolysis of triglycerides which are transported in plasma by chylomicrons and very low density lipoprotein (VLDL). Consequently, it is possible that LPL secretion by human monocytes is related to atherogenesis. As shown below, human monocytes and guinea pig alveolar macrophages in culture synthesize and secrete LPL. The purpose of this research is to study the significance of this macrophage LPL.Therefore I investigated the effect of lipoprotein on LPL activity of guinea pig alveolar macrophages.Monocytes were obtained from a normal healthy donor and were separated from whole blood by Ficoll-Hypaque. The lungs of guinea pig were washed 10-12 times with 0.9% NaCl and the washings were cultured. The enzyme activity was determined by a modification of the method of Schotz et al. The separation of the lipoprotein fractions was performed in a type RP-40 rotor by ultracentrifugation by the method of Havel et al.The rate of LPL secretion from human monocytes was low initially but increased with time in culture. During 14 days in culture, maximal activity was attained on the 12th day.On the other hand, guinea pig alveolar macrophages increased from the beginning. During 8 days in culture, maximal activity was attained on the 4th day. Next, we investigated the effect of lipoprotein on LPL activity of guinea pig alveolar macrophages. Alveolar macrophages were cultured for 2 days with either RPMI-1640 medium containing 10% lipoprotein deficient serum (LPDS) or that medium with the addition of VLDL, LDL, and HDL respectively in the concentrations of 300μg protein/ml. When VLDL was added to LPDS after one day, LPL activity was significantly reduced to 48.3% within 2 days of exposure when compared with LPDS controls. (p<0.02). A possible explanation for this phenomenon, is that LPL activity may be inhibited by apoprotein CIII of VLDL.

Maturation and secretion of lipoprotein lipase in cultured adipose cells. I. Intracellular activation of the enzyme.

The intracellular pathway and the activation of lipoprotein lipase have been examined in differentiated Ob17 cells. These adipose cells were previously shown to secrete lipoprotein lipase during exposure to heparin. Treatment of the cells with cycloheximide and heparin leads to enzyme depletion, as shown by activity measurement and immunofluorescence microscopy. The repletion phase has been studied in the presence of monensin or carbonyl cyanide m-chlorophenylhydrazone, ionophores known to affect the intracellular transport of membrane and secretory proteins. Monensin-treated cells synthesize fully active lipoprotein lipase. Under these conditions the antigen accumulates in the Golgi apparatus and the heparin-stimulated enzyme release is extensively reduced. Carbonyl cyanide m-chlorophenylhydrazone-treated cells do not contain any enzyme activity but show detectable antigen which accumulates in the endoplasmic reticulum. Competition for binding to immobilized anti-lipoprotein lipase antibodies of mature and endoplasmic reticulum-sequestered antigens is observed. Carbonyl cyanide m-chlorophenylhydrazone removal is rapidly followed by a transient burst of enzyme activity and a redistribution of the antigen in the different subcellular compartments. Therefore, the results show that the activation of lipoprotein lipase is an intracellular event taking place after the enzyme exits from the endoplasmic reticulum and before it reaches the trans-Golgi cisternae.

Synthesis and secretion of lipoprotein lipase by human monocyte-derived macrophages.

Human monocytes isolated from either defibrinated blood or buffy coat were shown to produce and secrete lipoprotein lipase during culture. The secretion occurred constitutively. Low levels of enzyme activity in the medium from freshly isolated cells increased with time of incubation, and maximal activity was attained after 9 days. The addition of heparin resulted in a substantial increase of enzyme activity in the culture medium. The optimal concentration of heparin was about 2 U/ml. The production of lipoprotein lipase was dependent on the presence of serum in the culture medium, and the optimal supplementation of serum was 25-50%.

モルモット肺胞マクロファージのLPL活性について(第IV報)

Macrophage may play an important role in the atherogenic process because they appear to be precursors of some arterial wall foam cells. Recently, it has been reported that human peripheral monocytes in culture synthesize and secrete lipoprotein lipase (LPL). Consequently, it is possible that LPL secretion by human monocytes is related to atherogenesis. As shown below, guinea pig alveolar macrophages in culture synthesize and secrete LPL. Therefore we investigated the effect of lipoprotein on LPL activity of guinea pig alveolar macrophages.Alveolar macrophages were cultured for 2 days with either RPMI-1640 medium containing 10% lipoprotein deficient serum (LPDS) or that medium with the addition of VLDL, LDL, and HDL respectively in the concentration of 300μg protein/ml.When VLDL was added to LPDS after one day, LPL activity was significantly reduced to 48.3 within 2 days of exposure when compared with LPDS controls (p<0.02).We believe there is a possible explanation for this phenomenon, LPL activity may be inhibited by apoprotein C-III of VLDL.

Cells of a human monocytic leukemia cell line (THP-1) synthesize and secrete apolipoprotein E and lipoprotein lipase

A human cell line established from a patient of an acute monocytic leukemia (THP-1) retained an ability to synthesize and secrete plasma apolipoprotein E like protein. The protein was identified with monospecific antibody raised against human plasma apolipoprotein E. The cells also secreted lipoprotein lipase (EC 3.1.1.34). The enzyme was characterized as lipoprotein lipase on the basis of the requirement of apolipoprotein C-II as an activator and the inhibition of its activity by sodium chloride. The secretion of both apolipoprotein E and lipoprotein lipase was markedly enhanced in the process of differentiation into macrophage-like cells by the addition of 4β-phorbol 12β-myristate 13α-acetate.

Uptake and degradation of human chylomicrons by macrophages in culture. Role of lipoprotein lipase.

Because macrophages secrete lipoprotein lipase (LPL), we sought to determine if LPL activity would influence the metabolism of chylomicrons by macrophages. In initial studies, we showed that normal chylomicrons were a substrate for the macrophage's LPL activity. Uptake of normal chylomicrons occurred in a saturable fashion and was effectively competed for by human chylomicrons, very low density lipoproteins (VLDL), and rabbit beta-VLDL, but not by acetyl-low density lipoprotein (LDL), and only modestly by native LDL. When apoprotein C-II-deficient chylomicrons were incubated with macrophages, no hydrolysis of triglyceride occurred, yet saturable uptake of chylomicron protein and lipid occurred, demonstrating that LPL activity is not a prerequisite for saturable uptake. However, addition of apo C-II led to marked hydrolysis and enhanced uptake of protein and lipid moieties. When albumin was present in the medium, there was approximately equal enhancement of cellular content of triglyceride and cholesteryl ester, despite the fact that chylomicrons are triglyceride-rich. This was due to uptake of a triglyceride-depleted particle produced by LPL, as well as a preferential re-release of triglyceride. These studies suggest potential pathways by which triglyceride-rich lipoproteins could contribute to accumulation of cholesteryl esters in macrophages, even while only small amounts of triglyceride accumulate.

The role of lipoprotein lipase in the metabolism of triglyceride-rich lipoproteins by macrophages.

We have previously shown that cultured macrophages secrete lipoprotein lipase (LPL) into the culture medium. The purpose of these experiments was to determine the role of LPL in the uptake of very low density lipoproteins (VLDL). Both J774 cells and mouse peritoneal macrophages took up and degraded normotriglyceridemic VLDL in a saturable manner. Uptake of VLDL was effectively competed for by rabbit beta-VLDL, human VLDL, but not by native LDL or acetyl LDL. LPL activity accelerated saturable uptake of VLDL but was not a prerequisite for it. This was most clearly seen in experiments with apo-C-II-deficient VLDL. Although no hydrolysis of VLDL triglyceride occurred, saturable uptake and degradation of the C-II-deficient lipoprotein occurred. However, addition of apo-C-II enhanced saturable uptake. Normal VLDL also promoted cellular accumulation of both triglycerides and cholesteryl esters. Accumulation of triglyceride occurred by uptake of intact VLDL particles, by uptake of a triglyceride-depleted particle produced by the action of LPL, and by the direct uptake of free fatty acids generated by the activity of LPL. In turn, the latter process was influenced by the amount of albumin in the medium capable of being an acceptor for free fatty acids. Finally, we have shown that when albumin is present in the medium, the macrophage is capable of mobilizing most of its stored triglyceride over 24 h, even as its cholesteryl ester content remains constant. These studies may be relevant to the ability of VLDL to promote macrophage accumulation of cholesteryl ester, even as triglyceride accumulation is minimized.

Established cell lines from rat adipose tissue that secrete lipoprotein lipase.

A number of cell lines derived from the stromal-vascular fraction of rat adipose tissue have been established that represent a variety of morphologic types. Despite their differing morphology, all of these cell lines secrete lipoprotein lipase in response to heparin. Because lipoprotein lipase secretion has been attributed to the presence of preadipocytes in the stromal-vascular fraction, we examined these cell lines for adipocyte conversion. None of the cell lines converted to adipocyte morphology when held at confluency or when exposed to media supplemented with high concentrations of fatty acid or very low density lipoproteins. These cell lines therefore do not seem to be preadipocytes, despite the presence of lipoprotein lipase. Among these cell lines are several that display the "cobblestone" morphology of endothelial cells, although they lack angiotensin-converting enzyme activity, reactivity with Factor VIII antibodies, and Weibel-Palade bodies. A number of authentic endothelial cells were found to be negative for lipoprotein lipase secretion. These data suggest that the "endothelial-like" cell lines established from adipose tissue are not endothelial cells.

Lipoprotein lipase secretion by human monocytes and rabbit alveolar macrophages in culture.

Human peripheral blood monocytes and rabbit alveolar macrophages secreted lipoprotein lipase during culture. Within hours after plating, lipoprotein lipase had accumulated in the culture medium of monocytes, and the rate of accumulation increased with time in culture. The initial rate of secretion of lipoprotein lipase by alveolar macrophages was higher than in monocytes but decreased after 8--10 hr to values similar to those expressed by monocytes cultured for the same length of time. The enzyme was characterized as lipoprotein lipase (triacylglycero-protein acylhydrolase, EC 3.1.1.34) on the basis of pH optimum (7.8--8.2 for monocytes, 8.1 for alveolar macrophages), dependence on apolipoprotein C-II for activity, inhibition by 0.3--0.5 M sodium chloride and protamine sulfate, and retention of a heparin-Sepharose gel. The expression of lipoprotein lipase secretion by human monocytes may have important implications with respect to the development of foam cells in the arterial wall during atherogenesis.

Lipoprotein lipase secretion by human monocyte-derived macrophages.

Human monocyte-derived macrophages in culture produced lipoprotein lipase. Although freshly isolated blood monocytes did not secrete much lipase activity, 1 d in culture was sufficient to trigger measureable enzyme production. During 3 wk in culture, maximal activity was attained after 7 d. At all times, the culture medium contained more enzyme activity than did a serum-heparin eluate or a detergent extract of the cell layer. The lipase activity was stimulated by serum and was inhibited by preincubation with antiserum to bovine lipoprotein lipase or when assayed at a high salt concentration. Furthermore, the enzyme bound to a heparin-Sepharose affinity column at physiological ionic strength. Cells cultured from a subject with primary lipoprotein lipase deficiency secreted no detectable enzyme. Since macrophages are prominent components of atherosclerotic lesions in man, their ability to synthesize and secrete lipoprotein lipase may be important to atherogenesis.

Human monocytes in culture synthesize and secrete lipoprotein lipase

Within the first day in culture, human monocytes begin to synthesize and secrete a triglyceride lipase. The designation of this activity as lipoprotein lipase is based upon: 1) a requirement of serum or apolipoprotein C-II for full activity; 2) inhibition by 1M NaCl or apolipoprotein C-III 2; 3) a pH optimum of 8; and 4) binding to endothelial cells that is releasable by heparin. The enzyme also exhibits immunological cross reactivity with antibody to purified bovine milk lipoprotein lipase as does human postheparin plasma lipoprotein lipase. Lymphocytes and polymorphonuclear leukocytes do not appear to contain this enzyme.