Apolipoprotein A-II/A-I ratio is a key determinant in vivo of HDL concentration and formation of pre-beta HDL containing apolipoprotein A-II.

Overexpression of human apolipoprotein A-II (apo A-II) in mice induced postprandial hypertriglyceridemia and marked reduction in plasma HDL concentration and particle size [Boisfer et al. (1999) J. Biol. Chem. 274, 11564-11572]. We presently compared lipoprotein metabolism in three transgenic lines displaying plasma concentrations of human apo A-II ranging from normal to 4 times higher, under ad libitum feeding and after an overnight fast. Fasting dramatically decreased VLDL and lowered circulating human apo A-II in transgenic mice; conversely, plasma HDL levels increased in all genotypes. The apo A-I content of HDL was inversely related to the expression of human apo A-II, probably reflecting displacement of apo A-I by an excess of apo A-II. Thus, the molar ratios of apo A-II/A-I in HDL were significantly higher in fed as compared with fasted animals of the same transgenic line, while endogenous LCAT activity concomitantly decreased. The number and size of HDL particles decreased in direct proportion to the level of human apo A-II expression. Apo A-II was abundantly present in all HDL particles, in contrast to apo A-I mainly present in large ones. Two novel findings were the presence of pre-beta migrating HDL transporting only human apo A-II in the higher-expressing mice and the increase of plasma HDL concentrations by fasting in control and transgenic mice. These findings highlight the reciprocal modifications of VLDL and HDL induced by the feeding-fasting transition and the key role of the molar ratio of apo A-II/A-I as a determinant of HDL particle metabolism and pre-beta HDL formation.

Cholesterol homeostatic mechanisms in transgenic mice with altered expression of apoproteins A-I, A-II and A-IV.

Our understanding of the in vivo metabolic functions of apoA-I and A-II has greatly advanced with the use of transgenic mice, but the physiological role of apoA-IV remains elusive. Both apoA-I and A-II are necessary for the structural stability of high-density lipoprotein (HDL). Structural differences exist between human and mouse A apoproteins because: i) human cholesterol ester transfer protein, lecithin cholesterol acyl transferase and phospholipid transfer protein interact better with human apoA-I; ii) human apoA-I and A-II, alone or in combination, form polydisperse instead of monodisperse HDL particles. Human apoA-II overexpression has highlighted its inhibitory effect on lipoprotein lipase and hepatic lipase, resulting in hypertriglyceridemia and concomitantly decreased HDL and apoA-I. After long-term challenge with an atherogenic diet, mice are less protected against lesion formation by human apoA-II, mouse apoA-II being overtly proatherogenic. On the other hand, human apoA-I confers great protection against lesion formation and causes reduction of preexisting lesions. Human apoA-IV is also protective, although the mechanisms by which this protection is achieved remain to be determined.

Transgenic animals with altered high-density lipoprotein composition and functions.

Our understanding of HDL metabolism in vivo has greatly advanced from studies with transgenic animals. Interactions between HDL apolipoproteins, transfer proteins, lipolytic enzymes and receptors modulate HDL size, particle number and fractional catabolic rate. The protective effect of HDL on atherosclerosis depends on the combined actions of HDL proteins and the metabolism of apo B-lipoproteins.

Overexpression of human apolipoprotein A-II in mice induces hypertriglyceridemia due to defective very low density lipoprotein hydrolysis.

Two lines of transgenic mice, hAIItg-delta and hAIItg-lambda, expressing human apolipoprotein (apo)A-II at 2 and 4 times the normal concentration, respectively, displayed on standard chow postprandial chylomicronemia, large quantities of very low density lipoprotein (VLDL) and low density lipoprotein (LDL) but greatly reduced high density lipoprotein (HDL). Hypertriglyceridemia may result from increased VLDL production, decreased VLDL catabolism, or both. Post-Triton VLDL production was comparable in transgenic and control mice. Postheparin lipoprotein lipase (LPL) and hepatic lipase activities decreased at most by 30% in transgenic mice, whereas adipose tissue and muscle LPL activities were unaffected, indicating normal LPL synthesis. However, VLDL-triglyceride hydrolysis by exogenous LPL was considerably slower in transgenic compared with control mice, with the apparent Vmax of the reaction decreasing proportionately to human apoA-II expression. Human apoA-II was present in appreciable amounts in the VLDL of transgenic mice, which also carried apoC-II. The addition of purified apoA-II in postheparin plasma from control mice induced a dose-dependent decrease in LPL and hepatic lipase activities. In conclusion, overexpression of human apoA-II in transgenic mice induced the proatherogenic lipoprotein profile of low plasma HDL and postprandial hypertriglyceridemia because of decreased VLDL catabolism by LPL.

The -700/-310 fragment of the apolipoprotein A-IV gene combined with the -890/-500 apolipoprotein C-III enhancer is sufficient to direct a pattern of gene expression similar to that for the endogenous apolipoprotein A-IV gene.

Spatial gene expression in the intestine is mediated by specific regulatory sequences. The three genes of the apoA-I/C-III/A-IV cluster are expressed in the intestine following cephalocaudal and crypt-to-villus axes. Previous studies have shown that the -780/-520 enhancer region of the apoC-III gene directs the expression of the apoA-I gene in both small intestinal villi and crypts, implying that other unidentified elements are necessary for a normal intestinal pattern of apoA-I gene expression. In this study, we have characterized transgenic mice expressing the chloramphenicol acetyltransferase gene under the control of different regions of the apoC-III and apoA-IV promoters. We found that the -890/+24 apoC-III promoter directed the expression of the reporter gene in crypts and villi and did not follow a cephalocaudal gradient of expression. In contrast, the -700/+10 apoA-IV promoter linked to the -500/-890 apoC-III enhancer directed the expression of the reporter gene in enterocytes with a pattern of expression similar to that of the endogenous apoA-IV gene. Furthermore, linkage of the -700/-310 apoA-IV distal promoter region to the -890/+24 apoC-III promoter was sufficient to restore the appropriate pattern of intestinal expression of the reporter gene. These findings demonstrate that the -700/-310 distal region of the apoA-IV promoter contains regulatory elements that, in combination with proximal promoter elements and the -500/-890 enhancer, are necessary and sufficient to restrict apoC-III and apoA-IV gene expression to villus enterocytes of the small intestine along the cephalocaudal axis.

Hepatic VLDL secretion of genetically obese Zucker rats is inhibited by a high-fat diet.

Hepatocytes from obese and lean Zucker rats adapted to a control (C) or a high-fat (HF) diet were prepared for the study of fatty acid (FA) uptake, partition between oxidation and esterification, and very low density lipoprotein (VLDL) production. A first 2-h kinetic study showed higher oleate uptake on a C diet by obese rat cells and an almost exclusive esterification to triacylglycerol (TG), VLDL secretion being 2.5-fold higher in obese rat cells and enhanced 1.4-fold in both genotypes in the presence of 0.7 mM oleate vs. 0.1 mM or no oleate. Fat feeding 1) decreased oleate uptake, esterification, incorporation into VLDL-TG, and mass VLDL-TG secretion and 2) abolished the VLDL-TG increase by 0.7 mM oleate. Similar but more pronounced effects were obtained in fat-fed lean animals. A second kinetic study using very short incubation times up to 1 h confirmed that fat feeding decreased oleate uptake and esterification, greatly stimulating its oxidation and production of acetoacetate (obese) or acetoacetate and beta-hydroxybutyrate (lean). Synthesis of lactate and pyruvate greatly decreased under HF feeding, remaining higher in obese rat cells. The drastic inhibition of labeled and total hepatic VLDL-TG secretion in obese and lean Zucker rats by the HF diet could be partly explained by decreased exogenous FA availability for VLDL-TG synthesis through its greater channeling toward oxidation and, indirectly, by the altered hepatocyte metabolic state.

Inhibition of hepatic very-low-density lipoprotein secretion in obese Zucker rats adapted to a high-protein diet.

The effect of a high-protein (HP) diet on hepatic very-low-density lipoprotein (VLDL) secretion was studied in obese and lean Zucker rats. With the control (C) diet, isolated hepatocytes from obese as compared with lean rats displayed higher uptake of [1-14C]oleate 0.7 mmol/L, 95% of which was esterified to glycerolipids; greater oleate incorporation into VLDL-triacylglycerol (TG); 2.6 times higher total VLDL-TG secretion; and 11-fold higher de novo fatty acid synthesis. Adaptation to HP feeding decreased weight gains in both phenotypes and hepatocyte TG content in obese rats. Oleate uptake by hepatocytes was appreciably reduced in the obese phenotype only. Despite esterification rates similar to those for the C diet, oleate incorporation into VLDL-TG decreased by 34% and 55% in obese and lean rats, respectively. Total (mass) VLDL-TG secretion was drastically decreased by 65% and 48% in obese and lean rat hepatocytes, respectively. HP feeding combined with overnight fasting accentuated the above decreases. Fatty acid synthesis was 50% lower in cells from HP-fed obese rats, but increased 1.7-fold in lean ones. Plasma glucagon increased in both phenotypes under HP feeding, whereas plasma insulin either increased (obese) or decreased (lean), with the insulin to glucagon ratio slightly decreasing. Thus, HP feeding drastically inhibited hepatic VLDL secretion in obese and lean Zucker rats by an undefined mechanism that was apparently related neither to de novo fatty acid synthesis nor to changes in oleate partitioning between esterification and oxidation.

Effect of a high-fat diet on the incorporation of stored triacylglycerol into hepatic VLDL.

Triacylglycerol (TG) stored in cytoplasmic lipid droplets of hepatocytes was labeled by in vivo [1-(14)C]oleic acid injection to study the effect of a high-fat diet on its incorporation into very-low-density lipoproteins (VLDL). Compared with the control diet, hepatocytes of fat-fed rats 1) contained 7.6 times more cytoplasmic (floating fat) TG and 1.9 times more endoplasmic reticulum (microsomal) TG; 2) had 8 and 6 times lower TG specific activities in cytoplasm and endoplasmic reticulum, respectively; 3) incorporated 22% less 14C label into hepatocyte esterified lipids (TG, cholesterol, phospholipid); 4) secreted 48 and 33% less radioactive and total VLDL-TG, respectively; 5) oxidized more cytoplasmic TG-fatty acid (FA); and 6) showed a 50% decreased total utilization of stored TG-FA. With both diets, the lysosomal inhibitor chloroquine concomitantly decreased productions of labeled VLDL-TG, CO2, and acid-soluble oxidation products. The decreased incorporation of stored TG into VLDL-TG appreciably contributes to the overall inhibition of hepatic VLDL secretion by fat feeding. It appears to be related to the decreased mobilization rate of stored TG and its increased channelling toward oxidation.

Contribution of cytoplasmic storage triacylglycerol to VLDL-triacylglycerol in isolated rat hepatocytes.

The cytoplasmic triacylglycerol (TG) storage pool of isolated hepatocytes was labelled in order to evaluate its incorporation into very low density lipoproteins (VLDL). Rats were injected with [1-14C]oleate 2 min prior to surgery and cell incubations began 90-100 min thereafter. In keeping with the equilibration of the two TG pools (in smooth endoplasmic reticulum, SER, and cytoplasm) in 120 min (Stein, Y. and Shapiro, B. (1959) Am. J. Physiol. 196, 1238-1241) the bulk of radioactive TG at time zero was in the cytoplasm and TG specific activities were similar in cytoplasm and SER. Radioactive and total VLDL-TG secretions were greatly inhibited after 80 min by chloroquine which is assumed to block lysosomal hydrolysis of cytoplasmic TG. When the SER-TG pool was labelled by addition of [1-14C]oleate in vitro, chloroquine affected neither [1-14C]oleate uptake and esterification nor its incorporation into VLDL-TG from 15-20 min until 80 min. After 100 min, when [1-14C]oleate-TG was transferred back from cytoplasm to SER, chloroquine began to decrease radioactive VLDL-TG output and by 210 min caused the same inhibition as under the in vivo labelling condition. These results are consistent with an inhibition by chloroquine of the lysosomal hydrolysis of cytoplasmic TG resulting in a blockage of their back transfer to SER membranes whereas other steps of VLDL production were not affected, at least up to 100 min. This study also showed that stored TG is a quantitatively important VLDL precursor, sustaining VLDL production for several hours in the absence of exogenous fatty acids.

Intestinal very-low-density lipoprotein secretion in the genetically obese Zucker rat.

The objectives of this study were to measure intestinal very-low-density lipoprotein (VLDL) production in obese Zucker rats and to assess an eventual effect of a high-fat diet. VLDL secretion was specifically inhibited by orotic acid, and intestinal VLDL output was measured following the Triton WR-1339 method. After a control diet, total VLDL secretion (without orotic acid) was 4.8 +/- 0.3 and 1.4 +/- 0.1 mg triacylglycerol/ml in obese and lean rats, respectively, decreasing by 30% in obese rats after fat-feeding. Intestinal VLDL production was similar in obese and lean rats fed the control diet (0.32 +/- 0.05 and 0.27 +/- 0.05 mg triacylglycerol/ml, respectively), increasing 2.5-fold after fat-feeding in both genotypes. Thus, intestine contributed 21 and 60% of total VLDL in lean but only 7 and 24% in obese rats with the control and high-fat diets, respectively. These results show that the intestine of obese Zucker rats does not contribute to their hypertriglyceridemia, suggesting that it originates solely from liver. Moreover, their intestinal VLDL production was stimulated by fat-feeding to the same extent as in lean animals.

[Effects of very-low-density lipoproteins on fatty acid synthesis and VLDL secretion by isolated hepatocytes]. / Effets des lipoprotéines très légéres sur la synthèse des acides gras et la sécrétion des VLDL par les hépatocytes isolés.

Hepatic VLDL production appears to be correlated with de novo fatty acid synthesis (lipogenesis). In this study lipogenesis was inhibited in vitro in order to establish an eventual direct effect on VLDL secretion. Hepatocytes from rats fed a standard diet were incubated with three triglyceride-rich lipoproteins: chylomicrons and VLDL obtained from rats fed a high-fat diet and VLDL obtained from rats fed a standard diet. The inhibition of lipogenesis (10 to 55%) was proportional to the concentration of the lipoproteins added. Chylomicrons and VLDL originating mainly from the intestine (prepared from fat-fed rats) inhibited lipogenesis more effectively than VLDL produced essentially by the liver (prepared from rats fed a standard diet). However the secretion of newly synthesized fatty acids in the medium did not decrease. When hepatocyte lipogenesis was inhibited by the addition of 1 mM oleic acid, total VLDL secretion (expressed as nmol of VLDL triglyceride/10(6) cells) was unchanged compared to control cells incubated without oleic acid. Our results suggest that hepatic VLDL secretion is not directly related to de novo fatty acid synthesis.

Oleate metabolism in isolated hepatocytes from lean and obese Zucker rats. Influence of a high fat diet and in vitro response to glucagon.

The uptake and metabolism of [1-14C]oleate (0.3 mmol/L) were studied in isolated hepatocytes from lean and obese Zucker rats fed either a control (low-fat) diet or a high-fat diet. With the control diet, [1-14C]oleate uptake was increased by 70% in the obese rats, and fat-feeding decreased this uptake to values comparable to that of their lean littermates. Interestingly, the hepatocyte mean surface area was increased in the obese mutants by 21% with the control diet and by 30% with the high-fat diet. The possible reasons for the differences in oleate uptake are discussed. With the control diet, cells from the obese rats showed a four-fold rise in [1-14C]oleate esterification, while ketogenesis (beta-hydroxybutyrate + acetoacetate production) as well as the radioactive acid-soluble products were greatly depressed. Production of CO2 was very low and similar in both groups of animals. Adaptation to the high-fat diet in the obese rats resulted in a reversal between esterification and oxidation of oleate: the latter became the major metabolic pathway as in the lean rats. The ketogenic capacity was greatly if not completely restored. In the lean animals, glucagon stimulated ketogenesis both in the presence or absence of oleate and decreased [1-14C]oleate esterification. In the obese rats, the hormone exerted a significant ketogenic effect only if oleate was present and did not influence its esterification. The data demonstrate the following abnormalities in the hepatocytes of obese Zucker rats: (1) an enlargement of cell size, (2) an increased oleate uptake, (3) a virtual absence of a ketogenic response to exogenous oleate, and (4) a markedly increased esterification of the latter. The metabolic defects, but not the cell size, appear to be largely corrected by an adaptation to a high-fat diet. The hepatic response to glucagon was decreased in the obese rats at the level of endogenous ketogenesis.

Intestinal very low density lipoprotein secretion in rats fed various amounts of fat.

1. The effect of a high-fat diet (30% fat by wt.) on intestinal very low density lipoprotein (VLDL) secretion was studied in male rats after specific inhibition of hepatic VLDL secretion by dietary orotic acid. Total VLDL secretion (from liver and intestine) was measured in animals not receiving orotic acid. 2. Fat-feeding resulted in a 32% decreased post-Triton secretion of total serum VLDL triacylglycerols as compared to a control (low fat) diet. Concomitantly, a large stimulation of post-Triton intestinal VLDL triacylglycerols secretion was measured in fat-fed rats. Thus, the major part (64%) of circulating triacylglycerols transported as VLDL originated from the intestine in these animals, leading presumably to an increased secretion of intestinal apolipoproteins. 3. Intestinal VLDL and chylomicron secretion rates increased with the amount of fat in the diet (7, 13, 20 or 30% fat by wt.). Whereas the chylomicron secretion was linearly related to the dietary fat content, the relationship between intestinal VLDL secretion and fat content of the diet was sigmoidal. The highest stimulation of intestinal VLDL formation was observed within a narrow range of dietary fat content (between 10 and 20%).

Very-low-density-lipoprotein secretion by isolated hepatocytes of fat-fed rats.

The very-low-density-lipoprotein secretion rate of isolated hepatocytes obtained from rats fed a high-fat diet was half that of cells from control animals. In fat-fed rats, the initial cellular uptake of [l-14C]oleate in vitro was decreased by 25%, its esterification to triacylglycerols and phospholipids by 50% and its incorporation into very-low-density-lipoprotein triacylglycerols by 70%. Exogenous oleate was not the main precursor of very-low-density lipoproteins in these animals. Lipogenesis, a minor source of very-low-density lipoproteins with the control diet in our experimental conditions, was inhibited by 84% after fat-feeding. A short-term inhibition of lipogenesis in vitro did not result in a decrease in very-low-density-lipoprotein secretion rate. The results suggest that fat-feeding decreased availability of exogenous as well as endogenous fatty acids for synthesis of very-low-density lipoproteins.

Effect of a high-fat diet on rat very low density lipoprotein secretion.

1. Very low density lipoprotein (VLDL) secretion rates were studied on rats adapted to a high-fat diet (71% calories as lard) for 3-4 weeks, compared to control (starch-fed) rats. 2. Experiments were performed at 14.00 h, at which time all animals had the same circulating free fatty acids. Fat-fed rats presented an apparent liver stealosis, a high post-Triton chylomicron secretion, but a 40% decreased VLDL secretion. 3. Injection of [1-14C]palmitic acid showed that the tracer was incorporated less in liver triacylglycerols of the fat-fed rats, presumably because of an enhanced ketogenesis. Secretion of labelled VLDL-triacylglycerols in 1 h was diminished 5-fold, even after a correction for the lower hepatic esterification. 4. Two complementary experiments were carried out, with the following results: at 08.00 h, when serum free fatty acid concentrations were comparable in both groups of rats [5,10], post-Triton VLDL secretion was diminished by 45% in the fat-fed rats; at 20.00 h, the fat-fed rats had significantly elevated plasma free fatty acids [5,10], but their VLDL secretion was the same as in control rats. 5. So it appears that in fat-fed rats circulating free fatty acids do not stimulate VLDL secretion as expected. It is suggested that the decreased VLDL secretion with the high-fat diet may result from inhibition of hepatic lipogenesis.

Diurnal variations of plasma lipoproteins and liver lipids in rats fed starch sucrose or fat.

The incidence of the dietary source of energy on lipid transport and accumulation was investigated over a full nycthemeral cycle in adapted rats fed ad libitum. Starch, sucrose and lard were compared. Lipoprotein composition of the plasma, liver and plasma lipids and insulinemia were analyzed every 3 hours over 24 hours. The pattern of VLDL concentration was dependent on the nature of the energetic substrate. Feeding starch resulted in a remarkable stability of lipoproteins, liver and plasma lipids, despite clearcut diurnal variations in plasma non esterified fatty acids, insulinemia and liver glycogen. In sucrose-fed rats VLDL rose to a sharp maximum in the post prandial period (9-12:00) and were totally cleared by 18:00. In fat-fed rats, HDL were elevated during the night, suggesting a possible stimulation of their synthesis by dietary fat in the intestine. LDL were constantly elevated with peak values at 21:00 while VLDL were very low, even at night, despite elevated levels of non-esterified fatty acids. It is concluded that, in animals adapted to a high fat-diet, a high level of circulating non esterified fatty acids is not sufficient to promote the synthesis of VLDL. The main regulating factor appears to be the intensity of hepatic lipogenesis which is stimulated by sucrose and inhibited by lard. No correlation was found between variations in plasma VLDL and insulinemia.

Weight and metabolic changes induced by low carbohydrate-high fat diets in man and in rat.

Weight loss and potential toxicity of low carbohydrate-high fat diets were examined in 8 volunteer medical students given either a high fat diet or a high carbohydrate diet for 15 days, as well as in 36 Sprague-Dawley rats fed for 5 weeks a series of low carbohydrate diets (less than 1%), varying in protein and lipid proportions. A weight loss occurred with the low carbohydrate-high fat diets; serum cholesterol level increased in both man and rat; plasma triglycerides rose in man. In rat, we found an increase in hepatic lipid levels as in plasma ketone and non-esterified fatty acid concentrations. These effects seemed to be related to the increase in lipid intake rather than the lack of carbohydrates.