Athina-Despina Kalopissis

Overexpression of Human Apolipoprotein A-II in Mice Induces Hypertriglyceridemia Due to Defective Very Low Density Lipoprotein Hydrolysis

Two lines of transgenic mice, hAIItg-δ and hAIItg-λ, 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 V max 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.

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.

Short-term adaptation of postprandial lipoprotein secretion and intestinal gene expression to a high-fat diet

Western diet is characterized by a hypercaloric and hyperlipidic intake, enriched in saturated fats, that is associated with the increased occurrence of metabolic diseases. To cope with this overload of dietary lipids, the intestine, which delivers dietary lipids to the body, has to adapt its capacity in lipid absorption and lipoprotein synthesis. We have studied the early effects of a high-fat diet (HFD) on intestinal lipid metabolism in mice. After 7 days of HFD, mice displayed normal fasting triglyceridemia but postprandial hypertriglyceridemia. HFD induced a decreased number of secreted chylomicrons with increased associated triglycerides. Secretion of larger chylomicrons was correlated with increased intestinal microsomal triglyceride transfer protein (MTP) content and activity. Seven days of HFD induced a repression of genes involved in fatty acid synthesis (FAS, ACC) and an increased expression of genes involved in lipoprotein assembly (apoB, MTP, and apoA-IV), suggesting a coordinated control of intestinal lipid metabolism to manage a high-fat loading. Of note, the mature form of the transcription factor SREBP-1c was increased and translocated to the nucleus, suggesting that it could be involved in the coordinated control of gene transcription. Activation of SREBP-1c was partly independent of LXR. Moreover, HFD induced hepatic insulin resistance whereas intestine remained insulin sensitive. Altogether, these results demonstrate that a short-term HFD is sufficient to impact intestinal lipid metabolism, which might participate in the development of dyslipidemia and metabolic diseases.

Opposite Effects of Plasma From Human Apolipoprotein A-II Transgenic Mice on Cholesterol Efflux From J774 Macrophages and Fu5AH Hepatoma Cells

Overexpression of human apolipoprotein A-II (hapo A-II) in transgenic mice (hAIItg mice) induced marked hypertriglyceridemia and low levels of plasma high density lipoprotein (HDL) with a high hapo A-II content. We sought to determine whether cholesterol efflux to plasma and HDL from these mice would be affected. In the Fu5AH cell system, plasma from hAIItg mice induced a markedly lower cholesterol efflux than did control plasma, in accordance with the dependence of efflux on HDL concentration. Moreover, HDLs from hAIItg mice were less effective acceptors than were control HDLs. In the J774 macrophage cell system, pretreatment with cAMP, which upregulates ATP binding cassette transporter 1, induced a marked increase in the efflux to hAIItg plasma as well as to purified hapo A-I and hapo A-II, whereas it had no effect on cholesterol efflux to control plasma. A strong positive correlation was established between percent cAMP stimulation of efflux and plasma hapo A-II concentration. The cAMP stimulation of efflux to hAIItg mouse plasma may be linked to the presence of pre-&bgr; migrating HDL containing hapo A-II. Thus, despite lower HDL and apolipoprotein A-I contents, the increased ability of plasma from hAIItg mice to extract cholesterol from macrophage-like cells may have an antiatherogenic influence.

Effect of a high-fat diet on rat very low density lipo-protein 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.

Apolipoprotein A-II is a key regulatory factor of HDL metabolism as appears from studies with transgenic animals and clinical outcomes

The structure and metabolism of HDL are linked to their major apolipoproteins (apo) A-I and A-II. HDL metabolism is very dynamic and depends on the constant remodeling by lipases, lipid transfer proteins and receptors. HDL exert several cardioprotective effects, through their antioxidant and antiinflammatory capacities and through the stimulation of reverse cholesterol transport from extrahepatic tissues to the liver for excretion into bile. HDL also serve as plasma reservoir for C and E apolipoproteins, as transport vehicles for a great variety of proteins, and may have more physiological functions than previously recognized. In this review we will develop several aspects of HDL metabolism with emphasis on the structure/function of apo A-I and apo A-II. An important contribution to our understanding of the respective roles of apo A-I and apo A-II comes from studies using transgenic animal models that highlighted the stabilizatory role of apo A-II on HDL through inhibition of their remodeling by lipases. Clinical studies coupled with proteomic analyses revealed the presence of dysfunctional HDL in patients with cardiovascular disease. Beyond HDL cholesterol, a new notion is the functionality of HDL particles. In spite of abundant literature on HDL metabolic properties, a major question remains unanswered: which HDL particle(s) confer(s) protection against cardiovascular risk?

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.

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%).

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.