An electron microscopic and functional study of very low density lipoproteins in intestinal lymph

Previous studies with fasting rats showed that the intestine produces endogenous very low density lipoproteins (VLDL) which resemble those in the plasma. Intestinal VLDL also were found to be important in lipid transport during absorption of saturated but not of unsaturated fat. These findings depended upon separations of a chylomicron-rich fraction (S(f) > 400) from VLDL (S(f) 20-400) by preparative ultracentrifugation methods based on particle flotation rates. The present studies correlate this method with electron microscopic measurement of lipoprotein particle size. Almost all intestinal lymph lipoprotein particles from fasting rats were less than 750 A in diameter, and could not be distinguished morphologically from plasma VLDL. Cholestyramine administration or bile diversion led to decreased lymph lipid output, correlating with marked reduction in VLDL. This supports the concept that lymph VLDL contain endogenous lipid which is reabsorbed from the intestinal lumen. During exogenous fatty acid absorption, lymph lipoprotein particle sizes were significantly smaller after administration of palmitate than after administration of linoleate, a finding consistent with ultracentrifugal evidence of the importance of VLDL in lipid transport during palmitate absorption. These studies fully confirm and extend earlier observations. Together, they show that the intestine is a source of endogenous VLDL in the fasting animal. In addition, significant quantities of exogenous lipid are transported in VLDL during palmitate absorption, whereas with linoleate absorption nearly all lipid is in chylomicrons. These findings indicate that the small intestine plays a role in lipoprotein metabolism which extends beyond the absorption of dietary fat.     

Secretion and uptake of nascent hepatic very low density lipoprotein by perfused livers from fed and fasted rats

Livers from fed or 24-hr fasted male rats were perfused in a recycling system. VLDL labeled with [1-14C]oleate (95% in triglyceride), produced in separate perfusions of livers from fed rats, was added to the medium as a pulse. Uptake of VLDL 14C-labeled triglyceride by livers from fasted rats was less than that from fed rats regardless of addition of oleate. During the interval in which radioactive triglyceride was taken up, the mass of triglyceride in the medium increased, indicative of the synthesis and net secretion of triglycerides. The rates of secretion of VLDL and uptake of VLDL were both more rapid in livers from fed rats in comparison to those from fasted animals. It was calculated that about 50% of the triglyceride synthesized and secreted by the liver was taken back by livers from fed rats. The VLDL from livers of fasted rats did not contain any apoE detectable by SDS gel electrophoresis or by radioimmunoassay when no fatty acid or 166 mumol of oleic acid was infused. In contrast, apoE comprised 6% of the VLDL apoprotein derived from perfusion of livers from fed animals in the absence of added fatty acid, and 20% when the fed livers were infused with 166 mumol of oleic acid. However, the net output (accumulation) of apoE by fasted liver was only two-thirds that from fed livers. When lipoprotein-free rat plasma containing apoE (4 mg/dl) was used in place of bovine serum albumin, the VLDL secreted by livers from either fed or fasted rats contained apoE and was taken up to a similar extent by such livers. These data suggested that the apoE of the d greater than 1.21 g/ml fraction was transferred to newly secreted VLDL which then stimulated uptake of the VLDL by livers from fasted rats. With further stimulation of secretion of VLDL triglyceride by infusion of 332 mumol of oleic acid/hr, the percent of apoE in the VLDL secreted by livers from fasted rats increased to 20%, which was similar to that of the VLDL produced by livers from fed rats when either 166 or 332 mumol/hr was infused. These data suggest a relationship between rates of hepatic secretion of VLDL (TG) and apoE, and the association of apoE with the secreted VLDL. During fasting, reduced secretion of both VLDL and apoE resulted in a VLDL particle that was considerably diminished in content of apoE and, therefore, that would be taken up by the liver at a reduced rate, in comparison to that observed in the fed animal.     

Effects of sucrose feeding and injection of lipid transfer protein on rat plasma lipoproteins

Sucrose feeding increased rat plasma very-low-density lipoprotein (VLDL) triacylglycerol concentration and decreased the cholesterol level in high-density lipoprotein (HDL). Gel filtration chromatography cholesterol profiles of both normal-fed and sucrose-fed plasma lipoproteins showed a small peak of VLDL and a large peak of HDL. Injection of a partially purified human lipid transfer protein preparation into normal-fed rats did not alter the concentration of cholesterol in either VLDL or HDL to a great extent, but there was a disappearance of the larger HDL particles. Injection of lipid transfer protein into sucrose-fed rats resulted in an overall 35% reduction in the concentration of HDL cholesterol, a more dramatic loss of larger HDL particles and a slight decrease in the mean particle size of the major HDL population.     

Intestinal lipoprotein synthesis in control and hypercholesterolemic rats

Previous studies in our laboratory have shown that very-low-density lipoproteins (VLDL) synthesized by the intestine of the diet-induced hypercholesterolemic rat are enriched in cholesteryl esters and unesterified cholesterol compared with intestinal VLDL from control rats. In these studies, we isolated and characterized nascent intestinal Golgi intermediate-density lipoproteins (IDL, d 1.006-1.040 g/ml) and studied isotope incorporation into apoliproteins of Golgi VLDL from control and hypercholesterolemic rats. IDL were triacylglycerol-rich lipoproteins but contained more cholesteryl ester and protein than the corresponding Golgi VLDL fractions. IDL from hypercholesterolemic rats were enriched in cholesteryl esters to a greater extent than IDL from control rats. The apolipoprotein patterns of IDL fractions were the same as those of intestinal Golgi VLDL, consisting of apolipoproteins (apo) B-48, A-I and A-IV. Time-course isotope incorporation curves for apo A-I and A-IV in Golgi VLDL were similar, but they differed from curves for apo B-48. None of these curves was markedly altered in the hypercholesterolemic rat. We conclude that the major effect of increased dietary cholesterol on intestinal lipoprotein biosynthesis is to increase the percentage of cholesteryl esters in Golgi lipoproteins. Dietary cholesterol does not alter the apolipoprotein composition of Golgi lipoproteins, nor does it have a significant effect on the pattern of isotope incorporation into apolipoproteins of Golgi VLDL. The effect of cholesteryl ester enrichment on the subsequent metabolism of these particles in the circulation and the effect of these particles on hepatic lipoprotein production remain to be determined.     

Hypolipidemic effect of intravenous pluronic L-81 in fasted rats treated with Triton WR-1339: possible inhibition of hepatic lipoprotein secretion

Pluronic L-81 (L-81), a non-ionic hydrophobic surfactant, is a powerful inhibitor of the secretion of lipid-transporting chylomicrons from intestinal epithelial cells to lymph. Since the other major organ that secretes lipoproteins into the circulation is the liver, whose principal lipid secretory product is very low density lipoprotein (VLDL), we tested the hypothesis that L-81 will also inhibit hepatic lipid secretion. Rats were fasted so that they had little lipid input from the intestine. We then administered Triton WR-1339 (tyloxapol) intravenously to block peripheral utilization of VLDL, causing plasma lipids to rise rapidly. Some animals were also given L-81 intravenously to test whether the L-81 would retard the tyloxapol-induced rise in plasma lipids. Administration of tyloxapol alone (250 mg/kg) increased plasma triglyceride, phospholipid and cholesterol concentrations considerably. Simultaneous administration of a small dose of L-81 (6 mg/kg) markedly reduced the rise in plasma triglyceride, particularly in the first hour (by 45%). L-81 also diminished the rise in plasma phospholipid and cholesterol, but to a lesser extent (30%). In the fasting rat, most of the plasma triglyceride is in VLDL; therefore, L-81 probably acts by decreasing the secretion of hepatic VLDL. Thus, Pluronic L-81 may be a useful tool for examining the secretion and metabolism of hepatic lipoproteins, in particular, VLDL.     

Effect of lipid transfer protein on plasma lipids, apolipoproteins and metabolism of high-density lipoprotein cholesteryl ester in the rat

The role of human plasma lipid transfer protein (LTP) in lipoprotein metabolism was studied in the rat, a species without endogenous cholesteryl ester and triacylglycerol transfer activity. Partially purified human LTP was injected intravenously into rats. The plasma activity was between 1.5- and 4-fold that of human plasma during the experiments. 6 h after the injection of LTP, a significant increase in serum apoB, and no significant changes in serum total cholesterol, free cholesterol, triacylglycerols, apoA-I, apoE, or apoA-IV were noted. Cholesterol was increased in very-low density and low-density lipoproteins (VLDL and LDL) and decreased in large-sized apoE-rich HDL. ApoA-I-containing particles with a size smaller than in normal rats were present in serum of LTP-treated rats. The mean diameter of HDL particles decreased and apoE, normally present on large-sized HDL, was present on smaller sized particles. The metabolic fate of cholesteryl ester, originally associated with HDL, was studied by injection of [3H]cholesteryl linoleyl ether-labelled apoA-I-rich HDL in the absence and in the presence of LTP. The disappearance of [3H]cholesteryl linoleyl ether, injected as part of apoA-I-rich HDL, from serum was increased in the LTP-treated rats; the t1/2 changed from 3.9 to 2.2 h, resulting in an increased accumulation of [3H]cholesteryl linoleyl ether in the liver. This can be explained by the redistribution of HDL [3H]cholesteryl linoleyl ether to VLDL and LDL in the presence of LTP, leading to the combined contribution of VLDL, LDL and HDL to the hepatic uptake. The present findings show profound effects of LTP on the chemical composition of HDL subspecies, the size of HDL and on the plasma turnover and hepatic uptake of cholesteryl esters originally present in apo A-I-rich HDL.     

Binding of intermediate density lipoproteins rich or poor in high molecular weight apolipoprotein B to rat liver membranes

Very low density lipoproteins rich or poor in high molecular weight apolipoprotein B (Bh-rich or Bh-poor VLDL, respectively) were prepared from rats fasted for 2 days and animals fasted and then refed for 2 days, respectively. Bh-rich or Bh-poor VLDL remnants (IDL) were also prepared by in vitro lipolysis of the corresponding VLDL preparations, and their apolipoprotein (apo) profile and lipid composition determined. Bh-rich IDL are richer in esterified cholesterol than Bh-poor IDL, but poorer in apoC and triglycerides. The binding of 125I-labeled Bh-rich IDL and 125I-labeled Bh-poor IDL to rat liver membranes was assessed by saturation-curve studies. Both types of IDL bound to high- and low-affinity sites on rat liver membranes. There were no significant differences between the binding of IDL produced from Bh-rich or Bh-poor VLDL to either the high- or low-affinity sites. However, by masking the low-affinity binding sites with saturating amounts of human high density lipoproteins 3 (HDL3), we were able to demonstrate that Bh-rich IDL bound to high-affinity binding sites with five times less affinity than Bh-poor IDL. These results show that saturating the low-affinity binding sites of rat liver membranes reveals differences in the binding abilities of lipoproteins to the high-affinity sites. Also, an analysis of apo and lipid compositions of the two types of IDL reveals that the apoBh contribution is likely to be responsible for differences in affinities of IDL for the high-affinity binding sites of rat liver membranes.     

Rat McA-RH7777 cells efficiently assemble rat apolipoprotein B-48 or larger fragments into VLDL but not human apolipoprotein B of any size

Studies of truncated apoB peptides in human subjects with familial hypobetalipoproteinemia, as well as of puromycin-generated spectra of nascent apoB peptides in rat and hamster liver, suggest that a minimum size is required for N-terminal fragments of apoB to be efficiently assembled into full-sized VLDL. We report here results of experiments undertaken to examine this phenomenon in greater detail by expressing individual carboxyl-truncated human apoB constructs in McArdle cells. Thus, apoB-29, -32, -37, -42, -47, -53, -70 and full length apoB-100 were transiently expressed in rat McA-RH7777 hepatoma cells, or human apoB-31 and apoB-53 were stably expressed in the same cells, and the secreted VLDL particles were characterized by kinetic gradient ultracentrifugal flotation. Calibration with rat plasma VLDL subfractions showed that about 90 and 50%, respectively, of lipoprotein particles containing endogenous rat B-100 and B-48 floated between fractions 2;-8 of the 11-fraction gradient. This corresponds to the normal VLDL diameter range of about 47 to 28 nm, with the remaining half of rat B-48 recovered as HDL particles in the 1.1 g/ml range. In contrast, regardless of their size, only 2;-5% of any of the truncated human apoB peptides expressed in these cells was recovered in the VLDL region of the gradient. The remaining 95+% of the lipoproteins were found as high density particles; as previously found in other systems the densities of the latter were inversely related to their peptide chain-length. Furthermore, transiently expressed full-length human apoB-100 was inefficiently secreted as VLDL by these cells, with the remainder appearing as LDL-sized particles. Thus, although we showed that McA-RH7777 cells secreted endogenous rat apoB as normal-sized VLDL, we found them unsuitable for our original purpose of using human apoB fragments to further define effects of apoB size on VLDL assembly. These cells appeared unable to efficiently use any size of human apoB for that process. Pulse-labeled untransfected McA-RH7777 cells chased in the presence of puromycin did, however, show a sharp decline in VLDL assembly efficiency for endogenous nascent rat apoB peptides shorter than B-48, similar to that originally found in normal rat liver.     

Intracerebroventricular administration of leptin-induced apoptosis in the rat small intestinal mucosa

The localization of leptin and leptin receptors in the stomach and small intestine has been reported. Their function is still unknown, although leptin is a hormone that regulates appetite and fat-related metabolism. The small intestine is one of the important organs for regulating metabolism. The purpose of the present study was to investigate whether leptin regulates apoptosis in the small intestinal mucosa. Intestinal apoptosis was evaluated by percent fragmented DNA, electrophoresis, TUNEL staining, and western blotting analysis of caspase-3. Mucosal apoptosis in the rat jejunum and ileum was evaluated at 0, 3, 6, 12, and 24 hrs after injection. Rats were tested after ad libitum feeding and 24-hr fasting to exclude the anorectic effect of leptin. Leptin was injected intraperitoneally (ip) at a dose of 200 microg/rat and infused into the rat third cerebroventricle (icv) at a dose of 8 microg/rat. Leptin at a dose of 8 microg/rat significantly induced intestinal apoptosis in the small intestine at 3 and 6 hrs after icv administration in both ad libitum feeding and 24-hr fasted rats. This increase in apoptosis was not attenuated by vagotomy. Intestinal apoptosis increased 12 and 24 hrs after ip injection of leptin at a dose of 200 microg/rat. The peak of the increase in apoptosis in icv rats appeared earlier than that in ip rats. Leptin induced jejunal and ileal mucosal apoptosis in the rat, indicating that leptin might control intestinal function through the regulation of intestinal apoptosis.     

Endotoxin promotes preferential periportal upregulation of VLDL secretion in the rat liver

Zonation affects liver parenchymal cell function and metabolism as well as nonparenchymal cell activation, but whether VLDL production is zonated has yet to be elucidated. Infection induces enhanced VLDL secretion by the liver. Ex vivo studies were undertaken to examine the liver heterogeneity for VLDL formation and secretion and their in vivo response to endotoxin. Highly pure periportal (PP) and perivenous (PV) hepatocytes were isolated from fasted lipopolysaccharide-treated, fasted, and fed rats. They were used to assess their capacity to release VLDL-apolipoprotein B (apoB) and lipid classes in relation to de novo lipid synthesis and the expression of genes crucial to VLDL production. Despite the common superior ability of PP hepatocytes for lipid release and zonal differences in lipid synthesis, zonated secretion of VLDL particles was observed in septic but not in normal fed or fasted livers. The endotoxin-induced apoB secretion was more accentuated in PP hepatocytes; this was accompanied by a preferential PP increase in apoB and microsomal triglyceride transfer protein mRNA levels, whereas lipogenesis indicators were, if anything, similarly modified in hepatocytes of either acinar origin. We conclude that PP and PV hepatocytes exhibited similar capabilities for VLDL formation/secretion in normal conditions; however, the endotoxic pressure did zonate periportally.     

Post-secretory acquisition of apolipoprotein E by nascent rat hepatic very-low-density lipoproteins in the absence of cholesteryl ester transfer

Previous work has shown that nascent hepatic very-low-density lipoproteins (VLDL) in the rat are biosynthesized without the obligatory co-factor (apolipoprotein C-II) for lipoprotein lipase-mediated hydrolysis of their core triacylglycerols. Upon secretion, apolipoproteins C-II and C-III are rapidly transferred to the particles from high-density lipoprotein (HDL) within the space of Disse and upon the entry into the plasma. Here we extend those studies to include observations on the apolipoprotein E content and lipid composition of nascent hepatic VLDL before and after exposure to plasma components. We have elected to use hepatic secretory vesicle VLDL rather than liver perfusate VLDL as truly representative of the nascent lipoproteins. Nascent VLDL from fed rats has an apolipoprotein B/E ratio of 6.6 ± 0.5, whereas that from fasted animals is 13.9 ± 2.3. Incubation of nascent VLDL from fed and fasted rats with d > 1.063 g/ml rat serum, HDL or the d > 1.21 g/ml fraction resulted in a mass transfer of apoliproprotein E to the VLDL such that the apolipoprotein B/E ratio decreased to at least that of serum VLDL (3.4 ± 0.3). The d > 1.21 g/ml fraction appeared to contain a species of apolipoprotein E which most actively transferred to VLDL. The acquisition of apolipoprotein E by nascent secretory vesicle VLDL was attended by a loss of phospholipids, particularly the C₄₀ (stearoylarachidonyl) molecular species, and an increase in the cholesterol-to-phospholipid ratio from 0.11 ± 0.01 to 0.18 ± 0.03. No evidence was obtained to suggest a simultaneous acquisition of cholesteryl esters upon incubation of nascent VLDL with VLDL-free serum. We conclude that nascent hepatic VLDL is modified after secretion by acquisition of apolipoproteins C-II, C-III and E with a concomitant loss of phospholipids.     

Human apolipoprotein A-II associates with triglyceride-rich lipoproteins in plasma and impairs their catabolism

Postprandial hypertriglyceridemia and low plasma HDL levels, which are principal features of the metabolic syndrome, are displayed by transgenic mice expressing human apolipoprotein A-II (hapoA-II). In these mice, hypertriglyceridemia results from the inhibition of lipoprotein lipase and hepatic lipase activities by hapoA-II carried on VLDL. This study aimed to determine whether the association of hapoA-II with triglyceride-rich lipoproteins (TRLs) is sufficient to impair their catabolism. To measure plasma TRL residence time, intestinal TRL production was induced by a radioactive oral lipid bolus. Radioactive and total triglyceride (TG) were rapidly cleared in control mice but accumulated in plasma of transgenic mice, in relation to hapoA-II concentration. Similar plasma TG accumulations were measured in transgenic mice with or without endogenous apoA-II expression. HapoA-II (synthesized in liver) was detected in chylomicrons (produced by intestine). The association of hapoA-II with TRL in plasma was further confirmed by the absence of hapoA-II in chylomicrons and VLDL of transgenic mice injected with Triton WR 1339, which prevents apolipoprotein exchanges. We show that the association of hapoA-II with TRL occurs in the circulation and induces postprandial hypertriglyceridemia.     

The catabolism of human and rat very low density lipoproteins by perfused rat hearts.

The catabolism of human and rat 125I-labelled very low density lipoproteins (VLDL) was compared by perfusing the lipoproteins through beating rat hearts. Triacylglycerol was removed from the VLDL to a greater extent than the protein moiety, leaving remnants containing relatively more apo-B and less apo-C. The change in apo-C content of the remnants correlated with the loss of triacylglycerol. The extent of removal of triacylglycerol from the rat and human VLDL was similar and in most cases appeared to saturate the heart lipoprotein lipase. The remnants were slightly smaller in size than the VLDL, and included particles which appeared to be partially emptied. In addition to remnants of d less than 1.019 g/ml, iodinated lipoproteins derived from rat and human VLDL were recovered at d 1.019-1.063 and 1.063-1.21 g/ml. The former contained largely cholesterol and cholesteryl esters, while phospholipids were the dominant lipid in the latter. An average of 40% of the 125I-labelled apoprotein lost from the VLDL was associated with the perfused hearts. Very little d 1.019-1.063 g/ml lipoprotein was produced from low (physiological) concentrations of rat VLDL, most of the lipoprotein being removed by the heart. However, lipoproteins of density 1.019-1.063 g/ml were formed from human VLDL at all concentrations in the perfusate, as well as from higher concentrations of the rat VLDL. Agarose gel filtration of lipoproteins following heart perfusion with human VLDL revealed large aggregates containing particles which resemble low density lipoproteins (LDL) in electron microscopic appearance and apoprotein composition, since they contain largely apo-B. These data suggest that at normal concentrations rat VLDL are almost completely catabolised and taken up by the heart without the formation of LDL, while LDL is produced from human VLDL at all concentrations.     

Lipoprotein size is a main determinant for the rate of hydrolysis by exogenous LPL in human plasma

LPL is a key player in plasma triglyceride metabolism. Consequently, LPL is regulated by several proteins during synthesis, folding, secretion, and transport to its site of action at the luminal side of capillaries, as well as during the catalytic reaction. Some proteins are well known, whereas others have been identified but are still not fully understood. We set out to study the effects of the natural variations in the plasma levels of all known LPL regulators on the activity of purified LPL added to samples of fasted plasma taken from 117 individuals. The enzymatic activity was measured at 25°C using isothermal titration calorimetry. This method allows quantification of the ability of an added fixed amount of exogenous LPL to hydrolyze triglyceride-rich lipoproteins in plasma samples by measuring the heat produced. Our results indicate that, under the conditions used, the normal variation in the endogenous levels of apolipoprotein C1, C2, and C3 or the levels of angiopoietin-like proteins 3, 4, and 8 in the fasted plasma samples had no significant effect on the recorded activity of the added LPL. Instead, the key determinant for the LPL activity was a lipid signature strongly correlated to the average size of the VLDL particles. The signature involved not only several lipoprotein and plasma lipid parameters but also apolipoprotein A5 levels. While the measurements cannot fully represent the action of LPL when attached to the capillary wall, our study provides knowledge on the interindividual variation of LPL lipolysis rates in human plasma.     

Intestinal lipoproteins in the rat with D-(+)-galactosamine hepatitis

D-(+)-galactosamine (GalN) induces severe reversible hepatocellular injury in the rat accompanied by lecithin: cholesterol acyltransferase (LCAT) deficiency, defective chylomicron (CM) catabolism, and accumulation of abnormal plasma lipoproteins (Lps), including discoidal high density lipoproteins (HDL). These abnormalities are presumed to result from hepatic injury alone, but the effect of GalN on intestinal Lps has not been studied. To assess possible effects on intestinal Lp formation and secretion, mesenteric lymph fistula rats were injected with GalN or saline. Twenty-four hours later a 2-hr fasting lymph sample was collected; this was followed by an 8-hr duodenal infusion of a lipid emulsion containing 17.7 mM [3H]triolein at 3 ml/hr. Fasting lymph and fat-infused lymph flow rates, 3H, triglyceride, and cholesterol output, residual 3H in intestinal lumen and mucosa, total 3H recovery, and d less than 1.006 g/ml Lp size and lipid composition were unchanged by GalN treatment, but d less than 1.006 g/ml Lps were depleted of apoE and C. Fat-infused lymph phospholipid (PL) output was higher in GalN rats due to PL-enriched d greater than 1.006 g/ml Lps. Electron microscopy of lymph and plasma LDL and HDL revealed spherical Lps in all samples. GalN plasma, fasting lymph, and fat-infused lymph also contained large abnormal LDL and discoidal HDL. Control lymph LDL and HDL did not differ in size from control plasma LDL and HDL. Control lymph LDL contained both apoB240K and B335K. However, spherical LDL and discoidal HDL in fasting lymph from GalN rats differed significantly in size from the corresponding plasma particles and became closer in size to the plasma particles with fat infusion. GalN lymph LDL contained only apoB240K and had a lower PL/CE than GalN plasma LDL. GalN fasting lymph HDL, depleted of apoC and having a PL/CE of 5, became enriched in apoE and the PL/CE increased to 10 with fat infusion to closely resemble GalN plasma HDL. GalN reduces apoE and C (mainly of hepatic origin) in d less than 1.006 g/ml gut Lps, which may contribute to the CM catabolic defect in GalN rats. Lymph LDL and HDL, especially in fasting lymph, may be partially gut-derived with increased filtration of plasma Lps into lymph with fat infusion. GalN fat-infused lymph HDL is enriched in apoE, but unable to transfer apoE to d less than 1.006 g/ml intestinal Lps. We conclude that GalN hepatitis is a model that allows study of intestinal Lps with normal lipid digestion and absorption in the face of severe hepatic injury and LCAT deficiency.     

Hepatocellular triglyceride synthesis and transfer to lipid droplets and nascent very low density lipoproteins

The transfer of triglyceride from sites of synthesis in the endoplasmic reticulum to cytoplasmic lipid droplets and nascent VLDL (very low density lipoproteins) in rat liver in vivo has been examined with [3H]glycerol, cell fractionation, and electron microscopy. Rates of mass transfer of newly synthesized triglyceride were estimated from the specific radioactivity of triglyceride present in microsomal membranes and the radioactivity observed in recipient triglyceride pools. Fasting decreased the transfer of triglyceride to nascent VLDL without affecting transfer to lipid droplets. Stimulation of triglyceride synthesis with 2-tetradecylglycidic acid (TDGA) increased transfer of triglyceride to nascent VLDL 5-fold, and to lipid droplets 14-fold, 1 hr after TDGA administration. Triglyceride transfer to nascent VLDL was increased 6-fold, and to lipid droplets 37-fold, above control rates 6 hr following TDGA treatment, indicative of saturation of triglyceride assembly into nascent VLDL and storage of excess triglyceride in lipid droplet reservoirs. These liver triglyceride pools were concurrently expanded and electron microscopy demonstrated more abundant VLDL particles in the endoplasmic reticulum together with a proliferation of lipid droplets in hepatocytes. TDGA progressively decreased hepatic sn-glycerol-3-phosphate in fasting rats while triglyceride synthesis increased, indicating that sn-glycerol-3-phosphate does not limit the rate of triglyceride synthesis in this metabolic state. Results implicate triglyceride transfer from endoplasmic reticulum membranes to nascent VLDL as a regulated determinant of hepatic VLDL assembly and VLDL triglyceride secretion in vivo.     

Circadian rhythm of ornithine decarboxylase activity in small intestine of fasted rats.

The aim of this study was to determine whether the circadian changes in ornithine decarboxylase (ODC) activity of different segments of the small intestine were governed by factors other than food intake. First, the effects of fasting on mucosal ODC activity were examined. The results indicate that mucosal ODC activity in 24 hr and 48 hr fasted rats decreased significantly compared with ad libitum-fed rats. Second, the circadian rhythm of mucosal ODC activity was characterized by measuring mucosal ODC activity in fasted rats at four time points (09:00, 15:00, 21:00, and 03:00 hr; light period: 06:00-18:00 hr). The results from this study indicate that there is a detectable baseline ODC activity in different segments of fasting intestine. In duodenum, mucosal ODC activity was highest at 15:00 hr (light period), a time at which the rat was normally not eating. In jejunum and ileum, mucosal ODC activity increased between 21:00 and 03:00 hr (dark period). The observation that small intestine exhibits a distinct circadian rhythm of ODC activity in fasted rats suggests that not only food but also intrinsic factors can modulate physiologic oscillations in mucosal ODC activity.     

The demonstration of very low density lipoprotein in the basal lamina of the granulosa layer in the hen's ovarian follicle.

The isolated basal lamina from the granulosa layer in ovarian follicles of the domestic fowl contains an abundance of spherical particles with a modal cross-sectional diameter of 25-30 nm. The lipid in this basal lamina is predominantly triacylglycerol and its total fatty acid composition resembles that of plasma very low density lipoprotein (VLDL). Immunodiffusion studies and immunoelectrophoresis indicated that this basal lamina contains diffusible antigen identifiable with plasma VLDL. Perfusion with an alkaline buffer displaced the particles from the basal lamina and subsequent perfusion with plasma VLDL in an acidic buffer resulted in the reappearance of particles of similar size and form. Alternatively, when the perfused basal lamina was subsequently perfused with VLDL-free serum, few particulate structures were observed. Measurements of total and VLDL triacylglycerol together with electron microscope studies of the untreated and of the perfused basal lamina provided further evidence for the identification of the majority of particles with plasma VLDL. Other particulate lipoprotein is most probably plasma low density lipoprotein (LDL). These studies demonstrated that this basal lamina is permeable to the circulating VLDL of the laying fowl.     

Metabolic fate of rat and human lipoprotein apoproteins in the rat

The fate of (125)I-labeled apolipoproteins was studied in vivo in rats that had received intravenous injections of (125)I-labeled rat HDL and (125)I-labeled human HDL, LDL, and VLDL. Plasma decay curves of rat and human HDL were exponential with similar half-lives in the circulation (11-12 hr). After injection, low molecular weight apolipoproteins (apoLP-alanine of human HDL and fraction HS-3 of rat HDL) were found to redistribute to other lipoproteins, predominantly VLDL. Decay curves of individual HDL proteins were constructed after lipoprotein fractionation, delipidation, and polyacrylamide gel electrophoresis. It was found that the half-lives of the different HDL apoproteins were not identical. A major rat HDL protein (52% of total counts) had a circulating half-life (t((1/2))) of 12.5 hr. Two others had a t((1/2)) of 8-9 hr while the t((1/2)) of several others was 11-12 hr. The t((1/2)) of three well-characterized human HDL apoproteins, apoLP-glutamine I, apoLP-glutamine II, and apoLP-alanine, were 13.5, 9.0, and 15.0 hr, respectively. The fate of (125)I-labeled human VLDL and LDL apoproteins in rats was similar to that described previously in humans. After injection of (125)I-labeled human VLDL into rats, apoLP-glutamic acid and apoLP-alanine rapidly transferred to rat HDL and were lost thereafter from the circulation from both VLDL and HDL. The apoLDL moiety of human VLDL moved metabolically to the LDL density range (d = 1.019-1.063) through a lipoprotein of intermediate density (d = 1.006-1.019).     

Effects of oleic acid on the biosynthesis of lipoprotein apoproteins and distribution into the very-low-density lipoprotein by the isolated perfused rat liver.

The effects of oleic acid on the biosynthesis and secretion of VLDL (very-low-density-lipoprotein) apoproteins and lipids were investigated in isolated perfused rat liver. Protein synthesis was measured by the incorporation of L-[4,5-3H]leucine into the VLDL apoproteins (d less than 1.006) and into apolipoproteins of the whole perfusate (d less than 1.21). Oleate did not affect incorporation of [3H]leucine into total-perfusate or hepatic protein. The infusion of oleate, however, increased the mass and radioactivity of the VLDL apoprotein in proportion to the concentration of oleate infused. Uptake of oleate was similar with livers from fed or fasted animals. Fasting itself (24 h) decreased the net secretion and incorporation of [3H]leucine into total VLDL apoprotein and decreased the output of VLDL protein by the liver. A linear relationship existed between the output of VLDL triacylglycerol (mumol/h per g of liver) and secretion and/or synthesis of VLDL protein. Net output of VLDL cholesterol and phospholipid also increased linearly with VLDL-triacylglycerol output. Oleate stimulated incorporation of [3H]leucine into VLDL apo (apolipoprotein) E and apo C by livers from fed animals, and into VLDL apo Bh, B1, E and C by livers from fasted rats. The incorporation of [3H]leucine into individual apolipoproteins of the total perfusate lipoprotein (d less than 1.210 ultracentrifugal fraction) was not changed significantly by oleate during perfusion of livers from fed rats, suggesting that the synthesis de novo of each apolipoprotein was not stimulated by oleate. This is in contrast with that observed with livers from fasted rats, in which the synthesis of the total-perfusate lipoprotein (d less than 1.210 fraction) apo B, E and C was apparently stimulated by oleate. The observations with livers from fed rats suggest redistribution of radioactive apolipoproteins to the VLDL during or after the process of secretion, rather than an increase of apoprotein synthesis de novo. It appears, however, that the biosynthesis of apo B1, Bh, E and C was stimulated by oleic acid in livers from fasted rats. Since the incorporations of [3H]leucine into the VLDL and total-perfusate apolipoproteins were increased in fasted-rat liver when the fatty acid was infused, part of the apparent stimulated synthesis of the VLDL apoprotein may be in response to the increased formation and secretion of VLDL lipid.