Overexpression of apoC-I in apoE-null mice: severe hypertriglyceridemia due to inhibition of hepatic lipase

Apolipoprotein C-I (apoC-I) has been proposed to act primarily via interference with apoE-mediated lipoprotein uptake. To define actions of apoC-I that are independent of apoE, we crossed a moderately overexpressing human apoC-I transgenic, which possesses a minimal phenotype in the WT background, with the apoE-null mouse. Surprisingly, apoE-null/C-I mice showed much more severe hyperlipidemia than apoE-null littermates in both the fasting and non-fasting states, with an almost doubling of cholesterol, primarily in IDL+LDL, and a marked increase in triglycerides; 3-fold in females to 260 +/- 80 mg/dl and 14-fold in males to 1409 +/- 594 mg/dl. HDL lipids were not significantly altered but HDL were apoC-I-enriched and apoA-II-depleted. Production rates of VLDL triglyceride were unchanged as was the clearance of post-lipolysis remnant particles. Plasma post-heparin hepatic lipase and lipoprotein lipase levels were undiminished as was the in vitro hydrolysis of apoC-I transgenic VLDL. However, HDL from apoC-I transgenic mice had a marked inhibitory effect on hepatic lipase activity, as did purified apoC-I. LPL activity was minimally affected. Atherosclerosis assay revealed significantly increased atherosclerosis in apoE-null/C-I mice assessed via the en face assay. Inhibition of hepatic lipase may be an important mechanism of the decrease in lipoprotein clearance mediated by apoC-I.     

Expression of human apolipoprotein A-II in apolipoprotein E-deficient mice induces features of familial combined hyperlipidemia

Familial combined hyperlipidemia (FCHL) is a common inherited hyperlipidemia and a major risk factor for atherothrombotic cardiovascular disease. The cause(s) leading to FCHL are largely unknown, but the existence of unidentified "major" genes that would increase VLDL production and of "modifier" genes that would influence the phenotype of the disease has been proposed. Expression of apolipoprotein A-II (apoA-II), a high density lipoprotein (HDL) of unknown function, in transgenic mice produced increased concentration of apoB-containing lipoproteins and decreased HDL. Here we show that expression of human apoA-II in apoE-deficient mice induces a dose-dependent increase in VLDL, resulting in plasma triglyceride elevations of up to 24-fold in a mouse line that has 2-fold the concentration of human apoA-II of normolipidemic humans, as well as other well-known characteristics of FCHL: increased concentrations of cholesterol, triglyceride, and apoB in very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL) and low density lipoprotein (LDL), reduced HDL cholesterol, normal lipoprotein lipase and hepatic lipase activities, increased production of VLDL triglycerides, and increased susceptibility to atherosclerosis. However, FCHL patients do not have plasma concentrations of human apoA-II as high as those of apoE-deficient mice overexpressing human apoA-II, and the apoA-II gene has not been linked to FCHL in genome-wide scans. Therefore, the apoA-II gene could be a "modifier" FCHL gene influencing the phenotype of the disease in some individuals through unkown mechanisms including an action on a "major" FCHL gene. We conclude that apoE-deficient mice overexpressing human apoA-II constitute useful animal models with which to study the mechanisms leading to overproduction of VLDL, and that apoA-II may function to regulate VLDL production.     

Severe hypertriglyceridemia in human APOC1 transgenic mice is caused by apoC-I-induced inhibition of LPL

Studies in humans and mice have shown that increased expression of apolipoprotein C-I (apoC-I) results in combined hyperlipidemia with a more pronounced effect on triglycerides (TGs) compared with total cholesterol (TC). The aim of this study was to elucidate the main reason for this effect using human apoC-I-expressing (APOC1) mice. Moderate plasma human apoC-I levels (i.e., 4-fold higher than human levels) caused a 12-fold increase in TG, along with a 2-fold increase in TC, mainly confined to VLDL. Cross-breeding of APOC1 mice on an apoE-deficient background resulted in a marked 55-fold increase in TG, confirming that the apoC-I-induced hyperlipidemia cannot merely be attributed to blockade of apoE-recognizing hepatic lipoprotein receptors. The plasma half-life of [3H]TG-VLDL-mimicking particles was 2-fold increased in APOC1 mice, suggesting that apoC-I reduces the lipolytic conversion of VLDL. Although total postheparin plasma LPL activity was not lower in APOC1 mice compared with controls, apoC-I was able to dose-dependently inhibit the LPL-mediated lipolysis of [3H]TG-VLDL-mimicking particles in vitro with a 60% efficiency compared with the main endogenous LPL inhibitor apoC-III. Finally, purified apoC-I impaired the clearance of [3H]TG-VLDL-mimicking particles independent of apoE-mediated hepatic uptake in lactoferrin-treated mice. Therefore, we conclude that apoC-I is a potent inhibitor of LPL-mediated TG-lipolysis.     

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.     

Effects of atorvastatin on fasting and postprandial complement component 3 response in familial combined hyperlipidemia

VLDL overproduction by enhanced hepatic FFA flux is a major characteristic of familial combined hyperlipidemia (FCHL). The postprandial complement component 3 (C3) response has been associated with impaired postprandial FFA metabolism in FCHL. We investigated the effects of 16 weeks of treatment with atorvastatin on postprandial C3 and lipid changes in 12 FCHL patients. Atorvastatin significantly lowered fasting plasma C3 and triglyceride (TG) in FCHL. Fasting TG and insulin sensitivity were the best predictors of fasting and postprandial C3. Postprandial triglyceridemia and C3 response, estimated as area under the curve (AUC), were significantly lowered by atorvastatin by 19% and 12%, respectively, albeit still elevated, compared with 10 matched controls. Postprandial FFA-AUC and postheparin plasma lipolytic activities remained unchanged after atorvastatin, suggesting no major effect on lipolysis. After atorvastatin, postprandial hydroxybutyric acid-AUC, which was elevated in untreated FCHL patients, was decreased, reaching values similar to those in controls. The present data show reduction of postprandial hepatic FFA flux in FCHL by atorvastatin, providing an additional mechanistic explanation for the reduction of VLDL secretion reported previously for atorvastatin. This was accompanied by a decrease in fasting plasma C3 concentrations and a blunted postprandial C3 response to an acute oral fat load.     

Comparison of maritime pine oil and fish oil effects on plasma lipoproteins in apolipoprotein E-deficient mice

The goal of this study was to compare the lipid lowering properties of maritime pine and fish oils in apolipoprotein E-deficient (KOE) mice, an animal model of hyperlipidemia. KOE mice were supplemented with either lard, fish or maritime pine oil (10% w/w) for one month. Compared to lard, fish and maritime pine oils decreased cholesterol (-31% and -52% respectively) and phospholipid (-41 and -52%) levels and increased triglyceride (+182% and +123%) levels. These lipid changes resulted in an enrichment in triglycerides and a depletion in cholesterol of VLDL+IDL plasma fraction as compared to lard-fed mice. These findings suggest that VLDL-triglyceride lipolysis is impaired in KOE mice fed fish or maritime pine oil.     

Apolipoprotein E participates in the regulation of very low density lipoprotein-triglyceride secretion by the liver

ApoE-deficient mice on low fat diet show hepatic triglyceride accumulation and a reduced very low density lipoprotein (VLDL) triglyceride production rate. To establish the role of apoE in the regulation of hepatic VLDL production, the human APOE3 gene was introduced into apoE-deficient mice by cross-breeding with APOE3 transgenics (APOE3/apoe-/- mice) or by adenoviral transduction. APOE3 was expressed in the liver and, to a lesser extent, in brain, spleen, and lung of transgenic APOE3/apoe-/- mice similar to endogenous apoe. Plasma cholesterol levels in APOE/apoe-/- mice (3.4 +/- 0.5 mM) were reduced when compared with apoe-/- mice (12.6 +/- 1.4 mM) but still elevated when compared with wild type control values (1.9 +/- 0.1 mM). Hepatic triglyceride accumulation in apoE-deficient mice was completely reversed by introduction of the APOE3 transgene. The in vivo hepatic VLDL-triglyceride production rate was reduced to 36% of control values in apoE-deficient mice but normalized in APOE3/apoe-/- mice. Hepatic secretion of apoB was not affected in either of the strains. Secretion of (3)H-labeled triglycerides synthesized from [(3)H]glycerol by cultured hepatocytes from apoE-deficient mice was four times lower than by APOE3/apoe-/- or control hepatocytes. The average size of secreted VLDL particles produced by cultured apoE-deficient hepatocytes was significantly reduced when compared with those of APOE3/apoe-/- and wild type mice. Hepatic expression of human APOE3 cDNA via adenovirus-mediated gene transfer in apoE-deficient mice resulted in a reduction of plasma cholesterol depending on plasma apoE3 levels. The in vivo VLDL-triglyceride production rate in these mice was increased up to 500% compared with LacZ-injected controls and correlated with the amount of apoE3 per particle. These findings indicate a regulatory role of apoE in hepatic VLDL-triglyceride secretion, independent from its role in lipoprotein clearance.     

A decreased expression of angiopoietin-like 3 is protective against atherosclerosis in apoE-deficient mice

KK/Snk mice (previously KK/San) possessing a recessive mutation (hypl) of the angiopoietin-like 3 (Angptl3) gene homozygously exhibit a marked reduction of VLDL due to the decreased Angptl3 expression. Recently, we proposed that Angptl3 is a new class of lipid metabolism modulator regulating VLDL triglyceride (TG) levels through the inhibition of lipoprotein lipase (LPL) activity. In this study, to elucidate the role of Angptl3 in atherogenesis, we investigated the effects of hypl mutation against hyperlipidemia and atherosclerosis in apolipoprotein E knockout (apoEKO) mice. ApoEKO mice with hypl mutation (apoEKO-hypl) exhibited a significant reduction of VLDL TG, VLDL cholesterol, and plasma apoB levels compared with apoEKO mice. Hepatic VLDL TG secretion was comparable between both apoE-deficient mice. Turnover studies revealed that the clearance of both [3H]TG-labeled and 125I-labeled VLDL was significantly enhanced in apoEKO-hypl mice. Postprandial plasma TG levels also decreased in apoEKO-hypl mice. Both LPL and hepatic lipase activities in the postheparin plasma increased significantly in apoEKO-hypl mice, explaining the enhanced lipid metabolism. Furthermore, apoEKO-hypl mice developed 3-fold smaller atherogenic lesions in the aortic sinus compared with apoEKO mice. Taken together, the reduction of Angptl3 expression is protective against hyperlipidemia and atherosclerosis, even in the absence of apoE, owing to the enhanced catabolism and clearance of TG-rich lipoproteins.     

In vivo evidence of defective postprandial and postabsorptive free fatty acid metabolism in familial combined hyperlipidemia

Overproduction of very low density lipoprotein (VLDL) is the major characteristic of subjects with familial combined hyperlipidemia (FCHL). As enhanced free fatty acid (FFA) flux to the liver may be one of the determinants of VLDL overproduction, we studied FFA changes and products of hepatic FFA metabolism in response to a 24-h oral fat loading test (50 g/m(2)) in 7 FCHL subjects and 7 matched control subjects. The response to the meal was subdivided into a postprandial (up to 8 h after ingestion of the meal) and postabsorptive period (from 8 to 24 h). Although postheparin plasma lipolytic activities were not different between both groups, the postprandial FFA area under the curve (FFA-AUC) and FFA incremental area under the curve (FFA-dAUC) were higher in FCHL subjects than in control subjects (6.05 +/- 0.45 vs. 3.43 +/- 0.46 and 2.60 +/- 0.49 vs. 0.96 +/- 0.31 mmol. h/L, respectively; P < 0.01 for each). The postprandial increase in ketone bodies was almost four times higher in FCHL patients. As ketogenesis occurs predominantly in hepatocytes, these findings suggest that during the postprandial period in FCHL an increased flux of FFA to the liver occurs, possibly because of inadequate incorporation of FFA into triglycerides (TGs) in adipocytes. In the postabsorptive period, FFA and ketone bodies significantly decreased in FCHL subjects, in contrast to control subjects, in whom both increased. These results may represent a diminished release of FFA from adipocytes by hormone-sensitive lipase (HSL) in FCHL patients. The decrease in postabsorptive FFA and ketone bodies in FCHL patients could not be explained by insulin-mediated inhibition of HSL, as both FCHL subjects and control subjects had similar postabsorptive insulin concentrations, which were below fasting concentrations.This study provides in vivo evidence of impaired metabolism of postprandial FFA in FCHL, which may explain in part the hepatic VLDL overproduction characteristic of FCHL subjects.     

Acute inhibition of hepatic lipase and increase in plasma lipoproteins after alcohol intake

Chronic alcohol intake is associated with an increase in fasting plasma high density lipoproteins (HDL). To study alcohol's acute effects on plasma lipoproteins, we measured plasma lipoprotein concentrations and activities of postheparin plasma lipases in nine normolipemic males after ingestion of 40 g of ethanol (as whiskey). After alcohol there was no change in lipoprotein lipase activity but hepatic lipase was decreased to 67% of baseline at 6 hr. There were associated increases in HDL phospholipids (12 mg/dl) and cholesterol (10 mg/dl) resulting in prominence of larger, lipid-enriched HDL particles. Changes were most pronounced in the HDL3 and HDL2a subclasses. Very low density lipoprotein (VLDL) phospholipids and cholesterol were also increased by 13 and 9 mg/dl, respectively, with no significant change in triglycerides. Changes in lipoproteins and lipase were largely reversed 10 hr after alcohol intake. The transient increases in VLDL and HDL lipids after alcohol may result in part from acute inhibition of hepatic lipase activity. The results suggest a role of hepatic lipase in the catabolism of phospholipids of VLDL and possibly HDL.     

Hormone-sensitive lipase deficiency in mice changes the plasma lipid profile by affecting the tissue-specific expression pattern of lipoprotein lipase in adipose tissue and muscle.

Hormone-sensitive lipase (HSL) is believed to play an important role in the mobilization of fatty acids from triglycerides (TG), diglycerides, and cholesteryl esters in various tissues. Because HSL-mediated lipolysis of TG in adipose tissue (AT) directly feeds non-esterified fatty acids (NEFA) into the vascular system, the enzyme is expected to affect many metabolic processes including the metabolism of plasma lipids and lipoproteins. In the present study we examined these metabolic changes in induced mutant mouse lines that lack HSL expression (HSL-ko mice). During fasting, when HSL is normally strongly induced in AT, HSL-ko animals exhibited markedly decreased plasma concentrations of NEFA (-40%) and TG (-63%), whereas total cholesterol and HDL cholesterol levels were increased (+34%). Except for the increased HDL cholesterol concentrations, these differences were not observed in fed animals, in which HSL activity is generally low. Decreased plasma TG levels in fasted HSL-ko mice were mainly caused by decreased hepatic very low density lipid lipoprotein (VLDL) synthesis as a result of decreased NEFA transport from the periphery to the liver. Reduced NEFA transport was also indicated by a depletion of hepatic TG stores (-90%) and strongly decreased ketone body concentrations in plasma (-80%). Decreased plasma NEFA and TG levels in fasted HSL-ko mice were associated with increased fractional catabolic rates of VLDL-TG and an induction of the tissue-specific lipoprotein lipase (LPL) activity in cardiac muscle, skeletal muscle, and white AT. In brown AT, LPL activity was decreased. Both increased VLDL fractional catabolic rates and increased LPL activity in muscle were unable to provide the heart with sufficient NEFA, which led to decreased tissue TG levels in cardiac muscle. Our results demonstrate that HSL deficiency markedly affects the metabolism of TG-rich lipoproteins by the coordinate down-regulation of VLDL synthesis and up-regulation of LPL in muscle and white adipose tissue. These changes result in an "anti-atherogenic" lipoprotein profile.     

The VLDL receptor plays a major role in chylomicron metabolism by enhancing LPL-mediated triglyceride hydrolysis

The VLDL receptor (VLDLr) is involved in tissue delivery of VLDL-triglyceride (TG)-derived FFA by facilitating the expression of lipoprotein lipase (LPL). However, vldlr-/- mice do not show altered plasma lipoprotein levels, despite reduced LPL expression. Because LPL activity is crucial in postprandial lipid metabolism, we investigated whether the VLDLr plays a role in chylomicron clearance. Fed plasma TG levels of vldlr-/- mice were 2.5-fold increased compared with those of vldlr+/+ littermates (1.20 +/- 0.37 mM vs. 0.47 +/- 0.18 mM; P < 0.001). Strikingly, an intragastric fat load led to a 9-fold increased postprandial TG response in vldlr-/- compared with vldlr+/+ mice (226 +/- 188 mM/h vs. 25 +/- 11 mM/h; P < 0.05). Accordingly, the plasma clearance of [3H]TG-labeled protein-free chylomicron-mimicking emulsion particles was delayed in vldlr-/- compared with vldlr+/+ mice (half-life of 12.0 +/- 2.6 min vs. 5.5 +/- 0.9 min; P < 0.05), with a 60% decreased uptake of label into adipose tissue (P < 0.05). VLDLr deficiency did not affect the plasma half-life and adipose tissue uptake of albumin-complexed [14C]FFA, indicating that the VLDLr facilitates postprandial LPL-mediated TG hydrolysis rather than mediating FFA uptake. We conclude that the VLDLr plays a major role in the metabolism of postprandial lipoproteins by enhancing LPL-mediated TG hydrolysis.     

Fatty acid cycling in the fasting rat

Adipose tissue lipolysis and fatty acid reesterification by liver and adipose tissue were investigated in rats fasted for 15 h under basal and calorigenic conditions. The fatty acid flux initiated by adipose fat lipolysis in the fasted rat is mostly futile and is characterized by reesterification of 57% of lipolyzed free fatty acid (FFA) back into adipose triglycerides (TG). About two-thirds of FFA reesterification are carried out before FFA release into plasma, whereas the rest consists of plasma FFA extracted by adipose tissue. Thirty-six percent of the fasting lipolytic flux is accounted for by oxidation of plasma FFA, whereas only a minor fraction is channeled into hepatic very low density lipoprotein-triglycerides (VLDL-TG). Total body calorigenesis induced by thyroid hormone treatment and liver-specific calorigenesis induced by treatment with beta, beta'-tetramethylhexadecanedioic acid (Medica 16) are characterized by a 1.7- and 1.3-fold increase in FFA oxidation, respectively, maintained by a 1.5-fold increase in adipose fat lipolysis. Hepatic reesterification of plasma FFA into VLDL-TG is negligible under both calorigenic conditions. Hence, total body fatty acid metabolism is regulated by adipose tissue as both source and sink. The futile nature of fatty acid cycling allows for its fine tuning in response to metabolic demands.     

Apolipoprotein AV accelerates plasma hydrolysis of triglyceride-rich lipoproteins by interaction with proteoglycan-bound lipoprotein lipase

Apolipoprotein A5 (APOA5) is associated with differences in triglyceride levels and familial combined hyperlipidemia. In genetically engineered mice, apoAV plasma levels are inversely correlated with plasma triglycerides. To elucidate the mechanism by which apoAV influences plasma triglycerides, metabolic studies and in vitro assays resembling physiological conditions were performed. In human APOA5 transgenic mice (hAPOA5tr), catabolism of chylomicrons and very low density lipoprotein (VLDL) was accelerated due to a faster plasma hydrolysis of triglycerides by lipoprotein lipase (LPL). Hepatic VLDL and intestinal chylomicron production were not affected. The functional interplay between apoAV and LPL was further investigated by cross-breeding a human LPL transgene with the apoa5 knock-out and the hAPOA5tr to an lpl-deficient background. Increased LPL activity completely normalized hypertriglyceridemia of apoa5-deficient mice; however, overexpression of human apoAV modulated triglyceride levels only slightly when LPL was reduced. To reflect the physiological situation in which LPL is bound to cell surface proteoglycans, we examined hydrolysis in the presence or absence of proteoglycans. Without proteoglycans, apoAV derived either from triglyceride-rich lipoproteins, hAPOA5tr high density lipoprotein, or a recombinant source did not alter the LPL hydrolysis rate. In the presence of proteoglycans, however, apoAV led to a significant and dose-dependent increase in LPL-mediated hydrolysis of VLDL triglycerides. These results were confirmed in cell culture using a proteoglycan-deficient cell line. A direct interaction between LPL and apoAV was found by ligand blotting. It is proposed, that apoAV reduces triglyceride levels by guiding VLDL and chylomicrons to proteoglycan-bound LPL for lipolysis.     

ApoC-III deficiency prevents hyperlipidemia induced by apoE overexpression

Adenovirus-mediated overexpression of human apolipoprotein E (apoE) induces hyperlipidemia by stimulating the VLDL-triglyceride (TG) production rate and inhibiting the LPL-mediated VLDL-TG hydrolysis rate. Because apoC-III is a strong inhibitor of TG hydrolysis, we questioned whether Apoc3 deficiency might prevent the hyperlipidemia induced by apoE overexpression in vivo. Injection of 2 x 10(9) plaque-forming units of AdAPOE4 caused severe combined hyperlipidemia in Apoe-/- mice [TG from 0.7 +/- 0.2 to 57.2 +/- 6.7 mM; total cholesterol (TC) from 17.4 +/- 3.7 to 29.0 +/- 4.1 mM] that was confined to VLDL/intermediate density lipoprotein-sized lipoproteins. In contrast, Apoc3 deficiency resulted in a gene dose-dependent reduction of the apoE4-associated hyperlipidemia (TG from 57.2 +/- 6.7 mM to 21.2 +/- 18.5 and 1.5 +/- 1.4 mM; TC from 29.0 +/- 4.1 to 16.4 +/- 9.8 and 2.3 +/- 1.8 mM in Apoe-/-, Apoe-/-.Apoc3+/-, and Apoe-/-.Apoc3-/- mice, respectively). In both Apoe-/- mice and Apoe-/-.Apoc3-/- mice, injection of increasing doses of AdAPOE4 resulted in up to a 10-fold increased VLDL-TG production rate. However, Apoc3 deficiency resulted in a significant increase in the uptake of TG-derived fatty acids from VLDL-like emulsion particles by white adipose tissue, indicating enhanced LPL activity. In vitro experiments showed that apoC-III is a more specific inhibitor of LPL activity than is apoE. Thus, Apoc3 deficiency can prevent apoE-induced hyperlipidemia associated with a 10-fold increased hepatic VLDL-TG production rate, most likely by alleviating the apoE-induced inhibition of VLDL-TG hydrolysis.     

ApoA-II maintains HDL levels in part by inhibition of hepatic lipase. Studies In apoA-II and hepatic lipase double knockout mice.

High density lipoprotein (HDL) cholesterol levels are inversely related to the risk of developing coronary heart disease. Apolipoprotein (apo) A-II is the second most abundant HDL apolipoprotein and apoA-II knockout mice show a 70% reduction in HDL cholesterol levels. There is also evidence, using human apoA-II transgenic mice, that apoA-II can prevent hepatic lipase-mediated HDL triglyceride hydrolysis and reduction in HDL size. These observations suggest the hypothesis that apoA-II maintains HDL levels, at least in part, by inhibiting hepatic lipase. To evaluate this, apoA-II knockout mice were crossbred with hepatic lipase knockout mice. Compared to apoA-II-deficient mice, in double knockout mice there were increased HDL cholesterol levels (57% in males and 60% in females), increased HDL size, and decreased HDL cholesteryl ester fractional catabolic rate. In vitro incubation studies of plasma from apoA-II knockout mice, which contains largely apoA-I HDL particles, showed active lipolysis of HDL triglyceride, whereas similar studies of plasma from apoA-I knockout mice, which contains largely apoA-II particles, did not. In summary, these results strongly suggest that apoA-II is a physiological inhibitor of hepatic lipase and that this is at least part of the mechanism whereby apoA-II maintains HDL cholesterol levels.     

Apolipoprotein B and triacylglycerol secretion in human triacylglycerol hydrolase transgenic mice

Apolipoprotein B (apoB)-containing lipoproteins play a critical role in whole body lipid homeostasis and the pathogenesis of atherosclerosis. The assembly of hepatic apoB-containing lipoproteins, VLDL, is governed by the availability of lipids, including triacylglycerol (TG). The majority of TG associated with VLDL is derived from the hepatic cytoplasmic lipid stores by a process involving lipolysis followed by reesterification. Microsomal triacylglycerol hydrolase (TGH) has been demonstrated to play a role in the lipolysis/reesterification process. To evaluate the potential regulatory role of TGH in hepatic VLDL assembly, we developed inducible transgenic mice expressing a human TGH minigene under the control of the mouse metallothionein promoter. Induction of human TGH by zinc resulted in liver-specific expression of the enzyme associated with 3- to 4-fold increases in lipolytic activity that could be attenuated with a TGH-specific inhibitor. Augmented TGH activity led to increased secretion of newly synthesized apoB and plasma TG levels. These results suggest that increased hepatic expression of TGH leads to a more proatherogenic plasma lipid and apoB profile.     

Resistance of a very low density lipoprotein subpopulation from familial dysbetalipoproteinemia to in vitro lipolytic conversion to the low density lipoprotein density fraction

In vitro lipolysis of very low density lipoprotein (VLDL) from normolipidemic and familial dysbetalipoproteinemic plasma by purified bovine milk lipoprotein lipase was studied using the combined single vertical spin and vertical autoprofile method of lipoprotein analysis. Lipolysis of normolipidemic plasma supplemented with autologous VLDL resulted in the progressive transformation of VLDL to low density lipoprotein (LDL) via intermediate density lipoprotein (IDL) with the transfer of the excess cholesterol to high density lipoprotein (HDL). At the end of 60 min lipolysis, 92-96% of VLDL triglyceride was hydrolyzed, and, with this process, greater than 95% of the VLDL cholesterol and 125-I-labeled VLDL protein was transferred from the VLDL to the LDL and HDL density region. When VLDL from the plasma of an individual with familial dysbetalipoproteinemia was substituted for VLDL from normolipidemic plasma, less than 50% of the VLDL cholesterol and 65% of 125I-labeled protein was removed from the VLDL density region, although 84-86% of VLDL triglyceride was lipolyzed. Analysis of familial dysbetalipoproteinemic VLDL fractions from pre- and post-lipolyzed plasma showed that the VLDL remaining in the postlipolyzed plasma (lipoprotein lipase-resistant VLDL) was richer in cholesteryl ester and tetramethylurea-insoluble proteins than that from prelipolysis plasma; the major apolipoproteins in the lipoprotein lipase-resistant VLDL were apoB and apoE. During lipolysis of normolipidemic VLDL containing trace amounts of 125I-labeled familial dysbetalipoproteinemic VLDL, removal of VLDL cholesterol was nearly complete from the VLDL density region, while removal of 125I-labeled protein was only partial. A competition study for lipoprotein lipase, comparing normolipidemic and familial dysbetalipoproteinemic VLDL to an artificial substrate ([3H]triolein), revealed that normolipidemic VLDL is clearly better than familial dysbetalipoproteinemic VLDL in competing for the release of 3H-labeled free fatty acids. The results of this study suggest that, in familial dysbetalipoproteinemic individuals, a subpopulation of VLDL rich in cholesteryl ester, apoB, and apoE is resistant to in vitro conversion by lipoprotein lipase to particles having LDL-like density. The presence of this lipoprotein lipase-resistant VLDL in familial dysbetalipoproteinemic subjects likely contributes to the increased level of cholesteryl ester-rich VLDL and IDL in the plasma of these subjects.     

Lipoprotein lipase deficiency and CETP in streptozotocin-treated apoB-expressing mice

Both hyperglycemia and hyperlipidemia have been postulated to increase atherosclerosis in patients with diabetes mellitus. To study the effects of diabetes on lipoprotein profiles and atherosclerosis in a rodent model, we crossed mice that express human apolipoprotein B (HuB), mice that have a heterozygous deletion of lipoprotein lipase (LPL1), and transgenic mice expressing human cholesteryl ester transfer protein (CETP). Lipoprotein profiles due to each genetic modification were assessed while mice were consuming a Western type diet. Fast-protein liquid chromatography analysis of plasma samples showed that HuB/LPL1 mice had increased VLDL triglyceride, and HuB/LPL1/CETP mice had decreased HDL and increased VLDL and IDL/LDL. All strains of mice were made diabetic using streptozotocin (STZ); diabetes did not alter lipid profiles or atherosclerosis in HuB or HuB/LPL1/CETP mice. In contrast, STZ-treated HuB/LPL1 mice were more diabetic, severely hyperlipidemic due to increased cholesterol and triglyceride in VLDL and IDL/LDL, and had more atherosclerosis.