Distribution of chylomicron degradation products in the isolated,perfused rat heart

Chylomicron degradation by hearts from fed and fasted rats was studied using a perfusion technique, which allows the separate collection of coronary (Q_rv) and interstitial effluent (Q_i). Upon perfusion with [³H]-cholesterol-containing chylomicrons the tissue recovery of label was highest in the fasted state, while label recovered in Q_i was highest in the fed state. Density gradient centrifugation of Q_i indicated that the label was recovered in lipoproteins with higher densities: low density lipoproteins (1.019<d<1.050), high density lipoproteins (1.050<d<1.21) and a fraction of d>1.21. These particles probably represent chylomicron degradation products (remnants and “surface fragments”). Our results indicate that tissue cholesterol uptake during chylomicron degradation may be inhibited in the fed state. Furthermore, the role of the myocyte (or interstitial) lipoprotein lipase in chylomicron degradation is discussed.     

Neutral lipase of rat heart: An inducible enzyme?

Post-nuclear supernatant (PNS) prepared from homogenates of heparin-pretreated adult rat hearts contains an acid and a neutral lipase activity. Both lipases preferentially hydrolyze endogenous PNS triglycerides (TG). PNS derived from newborn rat hearts, which is depleted of TG, lacks the neutral lipase activity. After dietary trierucate (TE)-induced cardiac lipidosis, the neutral lipase activity in PNS is markedly enhanced. TG-accumulation can also be induced upon $\text{in vitro}$ perfusion of rat hearts with Intralipid^® and rat serum. Intralipid^® -induced lipidosis is accompanied by an increased neutral lipase activity, which can be abolished when protein synthesis is inhibited by cycloheximide. Depletion of cardiac TG, during long-term perfusion, leads to a decrease in PNS neutral lipase activity. When PNS was prepared from hearts 5 h after cycloheximide pretreatment of rats the neutral lipase activities were reduced with a half-life of 6 h. Our data suggest that TG-mediated induction of neutral lipase synthesis is responsible for the increased rate of lipolysis observed during myocardial lipidosis.     

The relation between fatty acid mobilization and contractility in the isolated,perfused rat heart

The contractility of hearts from normal fed rats is decreased by 70% during perfusion with 50 μM chloroquine, which is a potent inhibitor of endogenous lipolysis. In triacylglycerol-rich hearts, obtained by feeding rats rapeseed-oil, chloroquine depresses lipolysis much less, while contractility was found to be inhibited only 30%. In both groups of hearts the effect of chloroquine was decreased by adding fatty acids, prostaglandin E₁, the $\text{Ca}{}^{\mathrm{2+}}\text{Mg}{}^{\mathrm{2+}}$ ionophore X-537A or more Ca²⁺ to the perfusion fluid. Norepinephrine and glucagon also stimulate chloroquine-depressed hearts. The conclusion is therefore reached that fatty acids act as Ca²⁺-vehicles in heart cells and that chloroquine, by inhibiting lipolysis, decreases Ca²⁺-transport by lowering unesterified fatty acid levels.     

Intracellular origin of hormone-sensitive lipolysis in the rat

The hormone-stimulated hydrolysis of endogenous triglycerides in heart and adipose tissue was found to be inhibited by chloroquine, which is known to accumulate in lysosomes and to inhibit the lysosomal degradation of protein and cholesterolesters.When the triglyceride depôt in heart cells was increased by feeding rats a diet enriched in erucic acid for three days prior to $\text{in vitro}$ perfusion of the heart, the spontaneous and the norepinephrine stimulated rates of lipolysis were both found to be increased. Both were inhibited by chloroquine. Analysis of trioleoylglycerol hydrolysis in heart homogenates after $\text{in vitro}$ heparin perfusion revealed the virtual absence of neutral lipase in contrast to an acid lipase activity. The results of this study suggest that lipolytic activity of lysosomal origin is the main source of hormone-sensitive endogenous triacylglycerol hydrolysis.     

On the metabolic function of heparin-releasable liver lipase

Intravenous administration of specific antibody against heparin-releasable liver lipase (liver lipase) induced a 75% inhibition of the enzyme activity $\text{in situ}$. Administration of the antibody resulted in an increase of high density lipoprotein (density range 1.050–1.13 g/ml; HDL₂) phospholipid levels (20% after 1 h; 54% after 4 h). Short-term (1 h) treatment with antibody had no significant effect on any of the other lipoprotein components. After long-term (4 h) treatment the free cholesterol level of HDL₂ and all components in the very low density lipoprotein (VLDL) + intermediate density lipoprotein (IDL) fraction were elevated (1.5–2.0 fold). In the low density lipoprotein (LDL) fraction only the phospholipid level was affected (increased by 72%). All lipid components in the HDL₃ fraction were decreased by the antibody treatment, but this decrease was only statistically significant for the cholesterolesters. The rate of removal of iodine-labeled high density lipoprotein (HDL) and LDL from serum was not affected by the antibody treatment.These results suggest that liver lipase may promote phospholipid removal $\text{in vivo}$ and show that a lowering of liver lipase $\text{in situ}$ has profound consequences for serum lipoprotein metabolism.     

Effect of glucose on AMP-catabolite release during fatty acid perfusion in normal and ischemic rat hearts

Isolated rat hearts were perfused retrogradely with a modified, oxygenated Tyrode solution containing 0.5 mM palmitate (complexed to albumin in a molar ratio of 6:1) with or without 11mM glucose. Fatty acid perfusion induced a decrease in contractile behaviour which was partly counteracted by glucose. The energy charge $\text{{([}\text{ATP}\text{] +}\text{1}\text{2}\text{[}\text{ADP}\text{])/([}\text{ATP}\text{] + [}\text{ADP}\text{] + [}\text{AMP}\text{]}}$ of the tissue was not altered although a significant drop was observed in creatine phosphate/ATP ratio in the absence of glucose. The release of AMP-catabolites, adenosine, inosine and hypoxanthine, occurring during fatty acid perfusion was reduced by glucose. In the absence of glucose fatty acids still induce lactate release indicating an enhanced glycogenolysis. In ischemic hearts the fatty acid-induced decrease in mechanical performance was significantly more severe when glucose was absent, while the glucose protection could also be observed in the energy charge of the ischemic tissue and the release of AMP-catabolites in the coronary effluent. The results suggest that loss of adenosine, inosine and hypoxanthine might contribute to the detrimental actions of a high fatty acid/albumin ratio upon the myocardium and confirms the protective action of glucose.     

The effect of α1-acid glycoprotein (orosomucoid) on triglyceride metabolism in the nephrotic syndrome

We have identified α₁-acid glycoprotein as a new co-factor in the lipoprotein lipase reaction. We isolated an active form of the compound from nephrotic urine that is effective both $\text{in vitro}$ and $\text{in vivo}$. α₁-acid glycoprotein increased lipolysis 100% in the presence of C-II apolipoprotein in a lipoprotein lipase assay system. Rats with induced nephrotic syndrome showed a decrease in triglyceride clearance. $\text{T}\text{1}\text{2}$ was increased from 14 min to 43 min. The injection of α₁-acid glycoprotein restored the lipid clearance to normal. These findings suggest that elevated plasma triglycerides in human nephrotic patients is the direct result of excessive loss of α₁-acid glycoprotein from plasma into urine. We propose that replacement therapy may be possible.     

Effectors of lipoprotein lipase secretion from isolated cardiac muscle cells incubated in vitro

Cycloheximide, colchicine, tunicamycin, glucagon, dibutyryl-3′–5′-cyclic AMP, dexamethasone and hydrocortisone had no effect on the lipoprotein lipase activity associated with rat cardiac muscle cells incubated $\text{in vitro}$. However, the steroid hormones and inhibitors affected profoundly the appearance of extracellular enzyme during the incubations. The pattern of effects, was consistent with lipoprotein lipase being a normal secretory product of heart muscle cells.     

Lipolysis of ApoC-II deficient very low density lipoproteins: enhancement of lipoprotein lipase action by synthetic fragments of apoC-II.

Enzymic hydrolysis of triacylglycerol has been studied with very low density lipoproteins from an individual with a genetically determined absence of apoC-II, the activator apoprotein for lipoprotein lipase. Normal rates of ester cleavage by purified bovine milk lipoprotein lipase can be achieved $\text{in}\text{vitro}$ with native apoC-II and by three shorter synthetic peptides, apoC-II(55–78), apoC-II(50–78) and apoC-II(43–78), which contain part of the carboxyl terminal third of the native apoprotein. At 0.5 μM concentration, all peptides produced a 7-fold activation. ApoC-II(43–78), but not apoC-II(50–78) or apoC-II(55–78), could bind VLDL as shown by separation of unbound ¹²⁵I peptides and the lipoproteins. Thus, residues 43–50 of apoC-II are part of a lipid binding region. High affinity binding of apoC-II peptides to the lipoprotein substrate is not obligatory for activation of lipoprotein lipase.     

The in vitro effect of growth hormone on adipose tissue lipoprotein lipase in rats

The $\text{in vitro}$ effect of growth hormone on adipose tissue lipoprotein lipase was studied in rats. Epididymal adipose tissue was incubated with human growth hormone in the presence of heparin. Growth hormone at a concentration of 0.1 μg per ml decreased by approximately 20% (p<0.005) the heparin-releasable lipoprotein lipase in rat adipose tissue. Discussion was focussed on the reciprocal changes caused by growth hormone of the activities of lipoprotein lipase and hormone-sensitive lipase in the rat adipose tissue.     

Early stimulation of alkaline phosphatase activity in response to 1 alpha, 25-dihydroxycholecalciferol.

Alkaline phosphatase activity (APA) stimulation in response to 1,25-dihydroxycholecalciferol (1,25 (OH)₂D₃) has been studied in vitamin-D-deficient rat intestinal brush borders prepared from $\text{ex}$-$\text{vivo}$-perfused duodeno-jejunal segments. Basal APA in intestines perfused with ethanol remained constant throughout the experiments. APA was significantly increased when intestines were perfused with 1,25 (OH)₂D₃ (3 nM) for 30, 45 or 60 min. A dose-effect response was observed when 1,25 (OH)₂D₃ increased in the perfusion medium. The maximal alkaline phosphatase activity after a 45-min perfusion (2404 ± 379 m^TU/mg prot.) was observed when 1,25 (OH)₂D₃ concentration was 6 nM. Cholecalciferol had no effect in this system.     

Hydrolysis of a fluorescent phospholipid substrate by phospholipase A2 and lipoprotein lipase

The fluorescent phospholipid 1-acyl-2-[6-[(7-nitro-2,1,3benzoxadiazol-4-yl)amino]-caproyl]phosphatidylcholine (C₆-NBD-PC) was used as a substrate for porcine pancreatic phospholipase A₂ (PA₂) and bovine milk lipoprotein lipase (LpL). Hydrolysis of C₆-NBD-PC by either enzyme resulted in a greater than 50-fold fluorescence enhancement with no shift in the emission maximum at 540 nm; Ca⁺⁺ was required for PA₂ catalysis. Identification of the products of hydrolysis showed cleavage at the $\text{sn}$-1 and $\text{sn}$-2 positions for LpL and PA₂, respectively. For PA₂, but not for LpL, there was a marked enhancement of enzyme catalysis at lipid concentrations above the critical micellar concentration of the lipid. Furthermore, apolipoprotein C-II, the activator protein of LpL for long-chain fatty acyl substrates, did not enhance the rate of catalysis of the water-soluble fluorescent phospholipid for either enzyme.     

Hormonal mediation of rat heart lipoprotein lipase activity after fat feeding

The effect of acute fat feeding on the response of two fractions of lipoprotein lipase in heart was explored. In rats, previously fasted, lipoprotein lipase activity released into the perfusate by heparin increased approximately 50% 4 h after fat feeding. The lipase activity remaining in the heart tissue after heparin perfusion showed no significant difference. When rats maintained ad libitum were intubated with glucose 2 h before the fat dose, a relatively larger increase (5-10-fold) in the heparin-releasable lipase activity was observed. The capacity of these hearts to hydrolyze 14C-labeled chylomicrons was also increased 4-5-fold over the controls. Fat ingestion has been reported to elevated plasma corticosteroid levels in rats. When adrenalectomized rats were fed fat, no significant changes in the heparin-releasable lipase activity were observed Hydrocortisone and corticotropin treatment increased the heparin-releasable lipase activity to the same degree as observed with fat feeding. These data suggest that the increase in heart lipoprotein lipase activity following fat feeding is mediated via corticosteroids.     

Acid- and neutral lipases involved in endogenous lipolysis in small intestine and heart

Trioleoylglycerol hydrolysis in homogenates of isolated small intestinal villus cells and hearts of rats showed pH optima at 5 and 7. The pH 7 enzyme(s) in contrast to the pH 5 enzyme could be inhibited by 1 μM diethyl $\text{p}$-nitrophenylphosphate. During vascular $\text{in vitro}$ perfusions of small intestine and heart this inhibitor severely depressed glycerol release. The lysosomotropic agent methylamine also inhibited endogenous lipolysis in these organs. It is concluded that both acid- and neutral lipases contribute to endogenous lipolysis in small intestine and heart.In heart evidence was obtained that an increase of the lipid depot, by previous trierucate feeding resulted in a relative increase of the contribution of the neutral lipase(s) to overall lipolysis. Extrapolation of this finding to adipose tissue, together with recent literature data, make it very likely that in this tissue neutral lipase activity is far more important than lysosomal enzyme activity in overall endogenous lipid degradation.     

Prostaglandins and cardiac anaphylaxis.

Manifestations of cardiac anaphylaxis include sinus tachycardia and arrhythmias, both of which result from histamine release. The marked decrease in coronary flow, which also occurs during cardiac anaphylaxis, cannot be attributed solely to histamine release.To indirectly assess the possible role of prostaglandins in cardiac anaphylaxis, hearts from sensitized guinea pigs were challenged $\text{in}$$\text{vitro}$ in the presence of indomethacin. This resulted in a marked increase in histamine release, which caused a greater tachycardia and an increase in the incidence of arrhythmias. Moreover, for the same degree of histamine release sinus rate increments were larger in the presence of indomethacin. However, despite the enhanced cardiac dysfunction, coronary flow rate did not decrease.The results suggest that, during cardiac anaphylaxis, prostaglandins modulate histamine release and the effects of released histamine. Furthermore, since we have found that PGF_2α is released from the anaphylactic heart, we tentatively ascribe the anaphylactic reduction in coronary flow to the elaboration of PGF_2α.     

On the nature of neutral lipase in rat heart

Neutral triacylglycerol lipase, which is not released by perfusion of rat hearts with heparin, is identical with lipoprotein lipase. The main criteria are 1) stimulation of neutral lipase by apolipoprotein C-II, 2) involvement of phospholipids in the hydrolysis of long-chain triacylglycerols, 3) alkaline shift of the pH activity curve by apolipoprotein C-II, 4) inhibition by protaminesulfate, 5) inhibition by an antibody against heparin-releasable lipoprotein lipase from heart and 6) binding of neutral lipase activity to Sepharose-bound heparin.The bulk of the non-releasable neutral lipase is not localized in the myocardiocytes, but in an extracellular compartment that is opened during Ca⁺⁺-free perfusion. The enzyme is probably involved in the uptake and not in the mobilization of lipid in the heart cells.     

Denovo synthesis of prolactin by human decidua

Relatively large amounts of immunoreactive prolactin were measured in homogenates of human decidual tissue obtained immediately after delivery of normal term pregnancies. In order to study the release and possible synthesis of prolactin by this tissue, explants of decidua were incubated for 24 hours at 37°C in oxygenated Gey's buffer containing 20% fetal calf serum. When cycloheximide was added to the medium in concentrations sufficient to prevent $\text{in}$$\text{vitro}$ protein synthesis, 85–90% of the prolactin present in the tissue was released into the medium during the first 3 hours of incubation. No additional prolactin accumulated in either the medium or the tissue during the remainder of the incubation period. In the absence of cycloheximide, the prolactin concentration in the medium increased progressively during incubation, so that after 24 hours the total amount of hormone present in the tissue and medium was significantly greater than that in the tissue and medium prior to incubation (37.6 ± 9.6 ng/ml at 0 time vs 82.2 ± 7.7 ng/ml at 24 hours). When ³H-1-leucine (100 u Ci) was supplied during incubation, radioactive proteins were detected in the medium at 24 hr, 14–20% of which were specifically precipitated by antiserum to human pituitary prolactin. When aliquots of this medium were chromatographed on Sephadex G-100, 80–95% of the ³H-proteins precipitated by antiserum to pituitary prolactin eluted in the same position as did purified, iodinated pituitary prolactin. These data indicate that a species of prolactin which is identical to pituitary prolactin by the criteria of immunoprecipitation and gel chromatography is synthesized by human decidual tissue $\text{in}$$\text{vitro}$.     

The mitogenic principle of Escherichia coli lipoprotein: B-lymphocyte mitogenicity of N-palmitoyl-cysteine and N-palmitoyl-glutamic acid α-methyl esters

N-Palmitoyl-cysteine methyl ester and N-palmitoyl-glutamic acid α-methyl ester, which are analogues to the lipophilic N-terminal part of the lipoprotein from the outer membrane of $\text{Escherichia coli}$, were synthesized and tested for biological activity in an $\text{in}\text{vitro}$ lymphocyte culture system: in spleen cells from several inbred mouse strains the fatty acid derivatives exhibited mitogenic activity towards B-lymphocytes comparable to the effect of lipoprotein, as measured by ³H-thymidine incorporation and by hemolytic plaque assays. These results confirm former investigations, which have shown that the mitogenic principle of the lipoprotein molecule resides in its N-terminal fatty acid-containing part. The proper dispersion of the water-insoluble substances was critical for their mitogenic activity. Optimal mitogenicity was obtained by sonicating the substances at concentrations of 0.1 mg/ml in culture medium.     

Contribution of microsomal cytochrome b5 electron transport system coupled with Δ5-3β-hydroxysteroid dehydrogenase to androgen synthesis from progesterone

Cytochromes P-450 and $\text{b}$₅ were observed in the microsomal fraction of interstitial tissue of rat testes. Microsomal cytochrome $\text{b}$₅ was reduced by the NADH coupled with the activities of Δ⁵-3β-hydroxysteroid dehydrogenase with Δ⁵-Δ⁴ isomerase through conversion of pregnenolone to progesterone. Activities of NADPH-supported 17α-hydroxylase and C-17-C-20 lyase which converted progesterone to androstenedione were stimulated by either the presence of NADH or the oxidative reaction by the dehydrogenase upon Δ⁵-3β-hydroxysteroids. Androstenedione production enhanced by the reaction of the dehydrogenase was decreased by addition of the antibody against NADH-cytochrome $\text{b}$₅ reductase which was purified from rat hepatic microsomes, suggesting the active participation of cytochrome $\text{b}$₅ in the androgen synthesis.     

Insulin-mediated modifications of myocardial lipoprotein lipase and lipoprotein metabolism

Recirculating organ perfusion in vitro was conducted with hearts from control rats, animals given a single dose of streptozotocin (65 mg/kg) 48 h earlier, and streptozotocin-treated rats administered insulin (5 units), 2 h prior to organ perfusion. During 45-min perfusions, the lipolysis of very low density lipoprotein (VLDL) triglyceride was significantly less in hearts from diabetics than in controls (41.9 +/- 7.3% of control). This was associated with significant reductions in heparin-releasable (functional) lipoprotein lipase and tissue lipoprotein lipase of perfused hearts. The decreases in VLDL triglyceride metabolism and the levels of myocardial lipoprotein lipase were completely reversed by treatment of diabetic rats with insulin 2 h prior to study. Similar improvement of VLDL triglyceride metabolism and increases in myocardial lipoprotein lipase activity were observed in hearts from diabetic rats by direct addition of 100 milliunits/ml of insulin to the recirculating perfusion media. Under these conditions, the increase in both fractions of lipoprotein lipase in response to insulin was completely inhibited, and utilization of VLDL triglyceride was partially inhibited by pre-perfusion with cycloheximide for 10 min. The data derived from either VLDL triglyceride lipolysis in organ perfusion or direct measurement of myocardial lipoprotein lipase demonstrate a direct effect of insulin on myocardial lipoprotein lipase activity, and suggest that the response to insulin may be due in part to effects on protein synthesis.