A biochemical and morphologic study of very low density lipoproteins in carbohydrate-induced hypertriglyceridemia.

On a high carbohydrate, fat-free diet, control and hypertriglyceridemic subject had a three-fold increase in d < 1.006, very low density lipoprotein (VLDL) triglyceride, and somewhat lesser increases in VLDL cholesterol and protein. Cholesterol and protein in 1.006 < d < 1.21 lipoprotein decreased in a reciprocal fashion, suggesting that these components might have been utilized in VLDL production. Electron microscope studies demonstrated a significant increase in the size of lipoprotein particles of the VLDL class and, in three of four subjects, an apparent increase in particle number. The change in particle size correlated with an increase in the triglyceride/protein ratio of the d < 1.006 lipoprotein. Hypertriglyceridemic individuals differed from the control subjects in that they had greater absolute increases in VLDL triglyceride, cholesterol, and protein, and greater decreases in 1.006 < d < 1.21 cholesterol and protein. In addition, they had larger VLDL particles with a higher triglyceride/protein ratio, both before the study and at the peak of the carbohydrate effect. The data suggest that the increase in plasma triglycerides induced by a high carbohydrate diet is usually due to the appearance in plasma of both greater numbers of VLDL particles and larger particles that are relatively richer in triglyceride content than those isolated during the basal state.

Diurnal variations of plasma lipids, tissue and plasma lipoprotein lipase, and VLDL secretion rates in the rat. A model for studies of VLDL metabolism.

Circadian rhythms of plasma lipids and lipoproteins, lipoprotein lipase activities and VLDL secretion rates were studied in fed and food-deprived (12 h) male rats after a light/dark synchronization of 14 days. In ad libitum fed rats, a circadian rhythm of plasma triacylglycerol, blood glucose and liver glycogen was clearly identified. A rhythm was also identified for plasma cholesterol, but not phospholipids. The peak of plasma triacylglycerol occurred 2 h after the beginning of the light period (7.00 a.m.), and the nadir, 2 h after the beginning of the dark period (7.00 p.m.). The differences of plasma triacylglycerol at these two circadian stages were even more pronounced in food-deprived rats and were confined to the very-low-density lipoprotein (VLDL) fraction. Plasma post-heparin and heart and muscle lipoprotein lipase activities were 50-100% higher at 7.00 p.m., the time when plasma triacylglycerol were lowest, as compared to 7.00 a.m. Plasma post-heparin hepatic lipase and adipose tissue lipoprotein lipase activities, in contrast, did not change. VLDL secretion rates were somewhat higher at 7.00 a.m. compared to 7.00 p.m., but this difference was not significant. It is concluded that physiological variation of heart and muscle lipoprotein lipase together with small differences of VLDL secretion rates are responsible for normal range oscillations of plasma VLDL triacylglycerol levels.

Origin and pattern of glucocorticoid-induced hyperlipidemia in rats. Dose-dependent bimodal changes in serum lipids and lipoproteins in relation to hepatic lipogenesis and tissue lipoprotein lipase activity.

Rats maintained for five days on a low dose of triamcinolone (0.5 mg/kg) showed a 2-fold increase in serum triacylglycerol concentration, paralleled by a rise in all very low density lipoprotein (VLDL) components but no significant change in serum cholesterol or high density lipoproteins (HDL). In contrast, a high dose of triamcinolone (12.5 mg/kg) produced a fall in triacylglycerol and VLDL to the range of control levels coincident with doubling in serum cholesterol and HDL. The rise in VLDL was attributed in a large part to enhanced hepatic fatty acid synthesis as evident from the marked rises in activity of rate-limiting enzymes of lipogenesis and in 3H incorporation into liver and serum fatty acids from in vivo administered 3H2O. The induction of fatty acid synthesis was linked to pronounced hyperinsulinemia, elicited by the triamcinolone treatment, to which the liver remained selectively responsive, contrary to the general insulin antagonism in peripheral tissues. Triamcinolone treatment also resulted in small rises in serum glucagon but these changes did not appear to be of importance for the observed bimodal serum lipoprotein perturbations. Dexamethasone, prednisolone and cortisol, administered in doses equipotent to 0.5 mg/kg triamcinolone, produced similar changes in the levels of serum triacylglycerol and insulin and activities of hepatic enzymes of lipogenesis.

Triacylglycerol metabolism and triacylglycerol lipase activities of cultured human skin fibroblasts.

The relative contribution of lipoproteins and free fatty acid to the cellular triacylglycerol content of cultured human fibroblasts was tested. Fibroblasts accumulated triacylglycerol in proportion to the molar ratio of free fatty acid (oleic acid) to albumin in the medium. Fibroblasts also accumulated triacylglycerol when exposed to medium containing human very low density liproprotein. This accumulation of triacylglycerol was apparently due to direct uptake of intact very low density lipoprotein particles initiated by binding of very low density lipoprotein to cell surface receptors. The amount of 125I-labeled very low density lipoprotein protein internalized and degraded by the cell saturated at the same very low density lipoprotein concentration that produced the maximum increase in cell triacylglycerol. Preincubations with lipoprotein-deficient serum, which enhanced the cell's ability to bind 125I-labeled very low density lipoprotein, increased the amount of 125I-labeled very low density lipoprotein internalized and degraded by the cell in parallel with increased levels of cellular triacylglycerol. Results suggest that the triacylglycerol that accumulates in the presence of very low density lipoprotein represents a lysosomal pool of partially degraded very low density lipoprotein. Measurements of lipase activity of fibroblast homogenates revealed three pH optima at (in descending order of magnitude of activity) pH 4, pH 6, and pH 8. The pH 8 lipase does not appear to represent lipoprotein lipase, since it is not activated by either serum or heparin. Exposure of the cells to medium with varying lipid composition had no effect on the lipase activities. The lipase activities of fibroblasts from donors with familial hypertriglyceridemia appear to be normal.

Effect of chorionic gonadotropin, triamcinolone, progesterone and estrogen on enzymes of placenta and liver in rats.

The activity of several enzymes of regulatory importance for the pathways of glycolysis, gluconeogenesis and lipogenesis was investigated in the placenta and liver of pregnant rats and in the liver of non-pregnant female rats. The rats received daily hormonal treatments on Days 15 to 17 of pregnancy and enzyme activities were measured on Day 18. Chorionic gonadotropin induced minor changes in enzyme activity, apart from a decrease in the activity of hepatic enzymes of lipogenesis in non-pregnant rats. Triamcinolone induced a marked increase in enzymes of gluconeogenesis and a decrease in the activity of pyruvate kinase in the liver of pregnant and non-pregnant rats; in contrast, inverse changes in activity, these enzymes were observed in the placenta. This response in the placenta was considered to arise not from direct hormone effect, but from the accompanying hyperglycemia and hyperinsulinemia. Triamcinolone also increased the activity of hepatic acetyl-CoA carboxylase in pregnant and non-pregnant rats, whereas it reduced the activity of this enzyme in the placenta. Estrogen produced changes similar to those of triamcinolone in the liver and placenta, except that it depressed the activity of acetyl-CoA carboxylase in both tissues. Progesterone had little effect on placental and hepatic enzymes. In general, the changes induced by these hormones in the placenta affected fewer enzymes than in the liver, were less extensive in magnitude and not necessarily in the same direction as in the liver. This indicates that the regulatory placental enzymes are subject to specific control mechanisms not necessarily influenced by direct hormone action.

Removal defect of very-low-density lipoproteins from diabetic rats.

The disappearance rate of triacyl[3H]glycerol carried on very-low-density lipoproteins (VLDL), isolated from diabetic rats and reinjected into normal recipient rats, was about twice as low as that of VLDL-triacyl[3H]glycerol from non-diabetic rats. The VLDL derived from diabetic rats was deficient in the apolipoprotein E component. These results indicate that the triacylglycerol removal defect in diabetes may be related to the quality of the protein carrier.

Hormone-responsive alkaline proteinase in rat skeletal muscle is not a mast cell-derived enzyme.

Proteinase activity was determined in myofibrils from intact rat skeletal muscle and from skeletal muscle myocytes grown in culture. In vivo administration of the mast cell degranulator compound 48/80 abolished the alkaline proteinase activity in myofibrils obtained from normal or streptozotocin-diabetic rats. Exposure of myocytes to compound 48/80 in cell cultures had no effect on their myofibrillar proteinase activity, nor did it affect the rate of overall protein degradation in these cells. Co-incubation of cultured mast cells (line P815Y) with myocytes followed by sonication of the cell mixture resulted in a marked reduction of the proteinase activity in the pellet fraction, suggesting that the mast cells contain inhibitor(s) of myofibrillar proteinase activity. It is suggested that the myofibril-bound alkaline proteinase activity is not a mast cell-derived enzyme but a genuine component of muscle cells. The in vivo 48/80-induced reduction of muscle myofibrillar proteinase activity appears to be due to release of a soluble inhibitory activity rather than removal of mast cell proteinase from the tissue by degranulation.

Kinetic and immunologic evidence for the absence of glucose-6-phosphatase in early human chorionic villi and term placenta.

The existence of the enzyme glucose-6-phosphatase (G6Pase) in early and term human placenta was investigated by comparing the characteristics of placental microsomal glucose 6-phosphate (G6P) hydrolytic activity and liver G6Pase. Placental microsomes exhibited similar apparent Km values for G6P and beta-glycerophosphate in intact and deoxycholate-treated microsomes, heat stability at acidic pH, low latency of mannose 6-phosphate hydrolysis, very low activity of pyrophosphate: glucose phosphotransferase, and undetectable [U-14C]G6P transport into the placental microsomes, all of which indicated that specific G6Pase activity does not exist in placenta. Immunological evidence of the absence of both 36.5 kDa and T2 proteins, which represent the G6Pase catalytic protein and the phosphate/pyrophosphate transporter protein, respectively, confirmed that early and term human placenta are devoid of the multicomponent G6Pase enzyme.

Experimental nephrotic syndrome: removal and tissue distribution of chylomicrons and very-low-density lipoproteins of normal and nephrotic origin.

Lymph chylomicrons and plasma VLDL, 14C-labelled in vivo, were isolated from normal and nephrotic rats and injected into normal or nephrotic recipients. In normal recipients, the half-life of chylomicrons of nephrotic vs. normal origin was significantly longer (5.2 +/- 0.5 vs. 3.5 +/- 0.4 min-1). The nephrotic chylomicrons were larger in size, deficient in apo-E and apo A-I, rich in triacylglycerol and cholesterol, but poor in phospholipids, indicating that factors related to composition affected their removal. The half-life of nephrotic vs. normal VLDL, given to normal recipients, was unexpectedly shorter, (4.5 +/- 0.2 vs. 5.8 +/- 0.2 min-1). The nephrotic VLDL were also triacylglycerol- and cholesterol-rich and phospholipid-poor, but had a large diameter spread and contained a dense fraction according to the zonal ultracentrifugation pattern, suggesting the presence of faster removable IDL-like particles. When nephrotic rats received normal particles, a pronounced removal delay was seen, paralleling the extent of plasma triacylglycerol elevation. The half-life of chylomicrons was 8.3 +/- 1.4 and 15.2 +/- 2.5 min-1 in moderately and severely nephrotic rats, respectively, that of VLDL was 11.72 +/- 2.1 and 37.8 +/- 7.1 min-1 correspondingly. The chylomicron-triacylglycerol uptake was reduced both by adipose tissues and muscles of normal or nephrotic recipients, with some increase in entry into lungs, kidneys and spleen. Tissue distribution patterns of VLDL-triacylglycerol was similar to that of chylomicrons, except that the liver took up approx. 90% of the label. The low share of triacylglycerol uptake by tissues rich in lipoprotein lipase indicates that the activity of this enzyme was unlikely to limit the rate of removal. Lipoprotein lipase activity in adipose tissue and heart was slightly decreased in moderately nephrotic rats and declined only by approx. 35% in severely nephrotic ones. These results indicate that the removal defect in nephrosis seems to be due, in part, to changes in the composition of triacylglycerol-rich particles, compromising their accessibility to lipolysis and, in part, to their abundance, saturating the lipolytic capacity.

Composition, removal and metabolic fate of chylomicrons derived from diabetic rats.

Tri[14C]acylglycerol-labelled chylomicrons, obtained from cannulated mesenteric lymph of streptozotocin-diabetic donor rats, when intravenously injected into non-diabetic recipient rats, disappeared from the circulation at a significantly slower rate than similarly prepared tri[14C]acylglycerol chylomicrons from non-diabetic donor rats (t1/2, 5.6 +/- 0.7 vs. 3.2 +/- 0.5 min-1, P less than 0.02). The appearance of labelled lipolysis products among plasma lipids (free fatty acid, cholesterol ester and phospholipid fractions) was delayed, indicating decreased availability for lipolysis of the chylomicron-borne triacylglycerol of diabetic origin. Tissue distribution of triacylglycerol, 15 min after the injection of chylomicrons to recipient rats, disclosed a 4-5-fold increase in uptake by muscles (heart and diaphragm) in relation to adipose tissues (epididymal and perirenal sites), in the case of chylomicrons of diabetic derivation. Since a large share of the chylomicron triacylglycerol was taken up by the liver, this tissue was perfused with chylomicron 'remnants' prepared by partial in vitro lipolysis with purified lipoprotein lipase. The 'remnants' of diabetic derivation were taken up by the liver at a 2-3-fold slower rate than those of non-diabetic origin. Chylomicrons derived from diabetic rats were found to be similar in size but markedly depleted of E apolipoproteins as determined by SDS-polyacrylamide gel electrophoresis, isoelectric focussing and a specific immunoassay. Decreases were also seen in A-I apolipoproteins by immunoassay and isoelectric focussing. Chylomicron 'remnants' were also markedly apolipoprotein E-deficient. In vitro incubation of the 'diabetic remnants' with high-density lipoproteins raised their apolipoprotein E content approx. 3-fold and considerably increased their hepatic uptake. Injection of intact chylomicrons preincubated with high-density lipoproteins likewise increased their in vivo removal rate toward the range of that of 'non-diabetic' chylomicrons. We conclude that diabetes-induced changes in the apolipoprotein composition of the chylomicrons and chylomicron remnants play an important role in their removal from the circulation. It appears that their recognition pattern is altered, reducing their ability to interact with receptor sites in the peripheral tissues and the liver, respectively.