Reduced intestinal fat absorptive capacity but enhanced susceptibility to diet-induced fatty liver in mice heterozygous for ApoB38.9 truncation

Fatty liver is prevalent in apolipoprotein B (apoB)-defective familial hypobetalipoproteinemia (FHBL). Similar to humans, mouse models of FHBL produced by gene targeting (apob(+/38.9)) manifest low plasma cholesterol and increased hepatic triglycerides (TG) even on a chow diet due to impaired hepatic VLDL-TG secretive capacity. Because apoB truncations shorter than apoB48 are expressed in the intestine, we examined whether FHBL mice may have limited capacity for intestinal dietary TG absorption. In addition, we investigated whether FHBL mice are more susceptible to diet-induced hepatic TG accumulation. Fat absorption capacity was impaired in apoB38.9 mice in a gene dose-dependent manner. Relative fractional fat absorption coefficients for apob(+/+), apob(+/38.9), and apob(38.9/38.9) were 1.00, 0.96, and 0.71, respectively. To raise hepatic TG, we fed high-fat (HF) and low-fat (LF) pellets. Hepatic TG level was observed in rank order: HF > LF > chow. On both LF and HF, liver TG level was higher in the apob(+/38.9) than in apob(+/+). Hepatic TG secretion remained impaired in the apob(+/38.9) on the HF diet. Thus the FHBL mice are more susceptible to diet-induced fatty liver despite relatively reduced intestinal TG absorption capacity on a HF diet.     

Hepatic overexpression of sterol carrier protein-2 inhibits VLDL production and reciprocally enhances biliary lipid secretion

We examined in vivo a role for sterol carrier protein-2 (SCP-2) in the regulation of lipid secretion across the hepatic sinusoidal and canalicular membranes. Recombinant adenovirus Ad.rSCP2 was used to overexpress SCP-2 in livers of mice. We determined plasma, hepatic, and biliary lipid concentrations; hepatic fatty acid (FA) and cholesterol synthesis; hepatic and biliary phosphatidylcholine (PC) molecular species; and VLDL triglyceride production. In Ad.rSCP2 mice, there was marked inhibition of hepatic fatty acids and cholesterol synthesis to <62% of control mice. Hepatic triglyceride contents were decreased, while cholesterol and phospholipids concentrations were elevated in Ad.rSCP2 mice. Hepatic VLDL triglyceride production fell in Ad.rSCP2 mice to 39% of control values. As expected, biliary cholesterol, phospholipids, bile acids outputs, and biliary PC hydrophobic index were significantly increased in Ad.rSCP2 mice. These studies indicate that SCP-2 overexpression in the liver markedly inhibits lipid synthesis as well as VLDL production, and alters hepatic lipid contents. In contrast, SCP-2 increased biliary lipid secretion and the proportion of hydrophobic PC molecular species in bile. These effects suggest a key regulatory role for SCP-2 in hepatic lipid metabolism and the existence of a reciprocal relationship between the fluxes of lipids across the sinusoidal and canalicular membranes.     

Short-term overexpression of DGAT1 or DGAT2 increases hepatic triglyceride but not VLDL triglyceride or apoB production

Increased triglyceride synthesis resulting from enhanced flux of fatty acids into liver is frequently associated with VLDL overproduction. This has led to the common belief that hepatic triglyceride synthesis can directly modulate VLDL production. We used adenoviral vectors containing either murine acyl-coenzyme A:diacylglycerol transferase 1 (DGAT1) or DGAT2 cDNA to determine the effect of a short-term increase in hepatic triglyceride synthesis on VLDL triglyceride and apolipoprotein B (apoB) production in female wild-type mice. Hepatic DGAT1 and DGAT2 overexpression resulted in 2.0-fold and 2.4-fold increases in the triglyceride content of liver, respectively. However, the increase in hepatic triglyceride content had no effect on the production rate of VLDL triglyceride or apoB in either case. Liver subfractionation showed that DGAT1 and DGAT2 overexpression significantly increased the content of triglyceride within the cytoplasmic lipid fraction, with no change in the triglyceride content of the microsomal membrane or microsomal VLDL. The increased cytoplasmic triglyceride content was observed in electron micrographs of liver sections from mice overexpressing DGAT1 or DGAT2. Overexpression of DGAT1 or DGAT2 resulted in enhanced [(3)H]glycerol tracer incorporation into triglyceride within cytoplasmic lipids. These results suggest that increasing the cytoplasmic triglyceride pool in hepatocytes does not directly influence VLDL triglyceride or apoB production. In the presence of adequate cytoplasmic lipid stores, factors other than triglyceride synthesis are rate-limiting for VLDL production.     

Isolation and characterization of lipoprotein of density less than 1.006 g/ml from rat hepatic lymph

The lipoprotein composition of rat hepatic lymph was studied using an animal model. The hepatic lymph duct of the adult male rat was cannulated and the hepatic lymph was collected. Hepatic lymph contains less than 1% of the total triglyceride output from the liver. Agarose gel electrophoresis of hepatic lymph showed the presence of two major lipoproteins bands, the alpha-migrating HDL and a band moving between plasma beta and pre-beta bands. Lipoprotein of density p less than 1.006 g/ml was then isolated by ultracentrifugation and it was found to correspond to the slow-moving pre-beta band. There was no difference in the mean diameter of hepatic lymph VLDL (64.4 nm) and that of plasma VLDL (64.6 nm). Compared with plasma VLDL, hepatic lymph VLDL has significantly more phospholipid (40% by weight), a higher cholesterol/cholesteryl ester ratio, and a marked reduction in triglyceride content (40% by weight). Although both plasma VLDL and hepatic lymph VLDL have apoE and apoB as the major apolipoproteins, there are other marked differences in apolipoprotein composition. Hepatic lymph VLDL has significantly less apoC and the apoB of hepatic lymph VLDL is predominantly the apoB240k (mol wt 240,000), with a small amount of the apoB330k (mol wt 335,000). On the other hand, plasma VLDL has an equal proportion of both apoB240k and apoB330k. This study presents for the first time the lipid and protein composition of rat hepatic lymph VLDL. Furthermore it has provided evidence that the hepatic lymph duct-cannulated rat can be used as an in vivo model for studying the secretion of nascent hepatic lymph VLDL.     

Cholesterol feeding strongly reduces hepatic VLDL-triglyceride production in mice lacking the liver X receptor alpha

The oxysterol-activated nuclear receptor liver X receptor alpha (LXRalpha) has been implicated in the control of both cholesterol and fatty acid metabolism. In this study, we have evaluated the effects of excess dietary cholesterol on hepatic cholesterol metabolism, lipogenesis, and VLDL production in homozygous (Lxralpha(-/-)), heterozygous (Lxralpha(+/-)), and wild-type mice. Mice were fed either chow or a cholesterol-enriched diet (1%, w/w) for 2 weeks. On the high-cholesterol diet, fractional cholesterol absorption was higher in Lxralpha(-/-) mice than in controls, leading to delivery of more dietary cholesterol to the liver. Lxralpha(-/-) mice were not able to induce expression of hepatic Abcg5/Abcg8, and massive accumulation of free cholesterol and cholesteryl esters (CEs) occurred. Interestingly, despite the inability to upregulate Abcg5/Abcg8, the highly increased hepatic free cholesterol content did stimulate biliary cholesterol output in Lxralpha(-/-) mice. Hepatic cholesterol accumulation was accompanied by decreased hepatic expression of lipogenic genes, probably caused by impaired sterol-regulatory element binding protein 1c processing, lower hepatic triglyceride (TG) contents, strongly reduced plasma TG concentrations (-90%), and reduced VLDL-TG production rates (-60%) in Lxralpha(-/-) mice. VLDL particles were smaller and CE-enriched under these conditions. Lxralpha deficiency did not affect VLDL formation under chow-fed conditions. Hepatic stearyl coenzyme A desaturase 1 expression was decreased dramatically in Lxralpha(-/-) mice and did not respond to cholesterol feeding, but fatty acid profiles of liver and VLDL were only slightly different between Lxralpha(-/-) and wild-type mice. Our data indicate that displacement of TGs by CEs during the VLDL assembly process underlies hypotriglyceridemia in cholesterol-fed Lxralpha(-/-) mice.     

A targeted apolipoprotein B-38.9-producing mutation causes fatty livers in mice due to the reduced ability of apolipoprotein B-38.9 to transport triglycerides

Nonphysiological truncations of apolipoprotein (apo) B-100 cause familial hypobetalipoproteinemia (FHBL) in humans and mice. An elucidation of the mechanisms underlying the FHBL phenotypes may provide valuable information on the metabolism of apo B-containing lipoproteins and the structure-function relationship of apo B. To generate a faithful mouse model of human FHBL, a subtle mutation was introduced into the mouse apo B gene by targeting embryonic stem cells using homologous recombination followed by removal of the selection marker gene by Cre-loxP-mediated site-specific recombination. The engineered mice bear a premature stop codon at residue 1767 and a 42-base pair loxP inserted into intron 24 of the apo B gene, thus closely resembling the apo B-38.9-producing mutation in humans. Apo B-38.9 was the sole apo B protein in homozygote (apob(38.9/38.9)) plasma. In heterozygotes (apob(+/)(38. 9)), apo B-100 and apo B-48 were reduced by 75 and 40%, respectively, and apo B-38.9 represented 20% of total circulating apo B. Hepatic apo B-38.9 mRNA levels were reduced by 40%. In cultured apob(+/)(38. 9) hepatocytes, apo B-100 was produced in trace quantities, and the synthesis rate of apo B-38.9 relative to apo B-48 was reduced by 40%. However, almost equimolar amounts of apo B-38.9 and apo B-48 were secreted into the media. Pulse-chase studies revealed that apo B-38. 9 was secreted at a faster rate and more efficiently than apoB-48. Nevertheless, both apob(+/)(38.9) and apob(38.9/38.9) mice had reduced hepatic triglyceride secretion rates and fatty livers. Thus, low mRNA levels or defective secretion of apo B-38.9 may not be responsible for the FHBL phenotypes caused by the apo B-38.9 mutation. Rather, a reduced capacity of apo B-38.9 for triglyceride transport may account for the fatty livers in these mice.     

Inhibition of ACAT by avasimibe decreases both VLDL and LDL apolipoprotein B production in miniature pigs.

An orally bioavailable acyl coenzyme A:cholesterol acyltransferase (ACAT) inhibitor, avasimibe (CI-1011), was used to test the hypothesis that inhibition of cholesterol esterification, in vivo, would reduce hepatic very low density (VLDL) apolipoprotein (apo) B secretion into plasma. ApoB kinetic studies were carried out in 10 control miniature pigs, and in 10 animals treated with avasimibe (10 mg/kg/d, n = 6; 25 mg/kg/d, n = 4). Pigs were fed a diet containing fat (34% of calories) and cholesterol (400 mg/d; 0.1%). Avasimibe decreased the plasma concentrations of total triglyceride, VLDL triglyceride, and VLDL cholesterol by 31;-40% 39-48%, and 31;-35%, respectively. Significant reductions in plasma total cholesterol (35%) and low density lipoprotein (LDL) cholesterol (51%) concentrations were observed only with high dose avasimibe. Autologous 131I-labeled VLDL, 125I-labeled LDL, and [3H]leucine were injected simultaneously into each pig and apoB kinetic data were analyzed using multicompartmental analysis (SAAM II). Avasimibe decreased the VLDL apoB pool size by 40;-43% and the hepatic secretion rate of VLDL apoB by 38;-41%, but did not alter its fractional catabolism. Avasimibe decreased the LDL apoB pool size by 13;-57%, largely due to a dose-dependent 25;-63% in the LDL apoB production rate. Hepatic LDL receptor mRNA abundances were unchanged, consistent with a marginal decrease in LDL apoB FCRs. Hepatic ACAT activity was decreased by 51% (P = 0.050) and 68% (P = 0.087) by low and high dose avasimibe, respectively. The decrease in total apoB secretion correlated with the decrease in hepatic ACAT activity (r = 0.495; P = 0.026).We conclude that inhibition of hepatic ACAT by avasimibe reduces both plasma VLDL and LDL apoB concentrations, primarily by decreasing apoB secretion.     

Regulation of hepatic secretion of very low density lipoprotein by dietary cholesterol.

Male rats were fed a cholesterol-free diet or the same diet supplemented with either 0.05, 0.1, 0.25, 0.5, 1, or 2% C for 21 days to investigate the effects of cholesterol on secretion of very low density lipoprotein (VLDL). Cholesterol feeding increased plasma and hepatic concentrations of triglyceride (TG) and cholesteryl esters (CE) in a dose-dependent manner. Plasma VLDL and low density lipoprotein (LDL) lipids were elevated by cholesterol feeding, while the high density lipoprotein (HDL) lipids were reduced. The secretion of the VLDL by perfused livers from these cholesterol-fed rats was examined to establish the relationship between the accumulation of lipids in the liver and the concurrent hyperlipemia. Liver perfusions were carried out for 4 h with a medium containing bovine serum albumin (3% w/v), glucose (0.1% w/v), bovine erythrocytes (30% v/v), and a 10-mCi 3H2O initial pulse. Oleic acid was infused to maintain a concentration of 0.6 mM. Hepatic secretion of VLDL-TG, PL (phospholipid), free cholesterol (FC), and CE increased in proportion to dietary cholesterol and was maximal at 0.5% cholesterol in these experiments in which TG synthesis was stimulated by oleic acid. Secretion of VLDL protein and apoB by the perfused liver was also increased. The molar ratios of surface (sum of PL and cholesterol) to core (sum of TG and CE) lipid components of the secreted VLDL, regardless of cholesterol feeding, were the same, as were the mean diameters of the secreted particles. The molar ratios of surface to core lipid of VLDL isolated from the plasma also were not affected by cholesterol feeding. During perfusion with oleic acid of livers from the rats fed the higher levels of cholesterol, the hepatic concentration of CE decreased, while the level of TG was not changed. We conclude that the hypercholesterolemia and hypertriglyceridemia that occur in vivo from cholesterol feeding, concurrent with accumulation of CE and TG in the liver, must result, in part, from increased hepatic secretion of all VLDL lipids and apoB. The VLDL particles produced by the liver of the cholesterol-fed rat are assembled without modification of the surface lipid ratios (PL/FC), but contain a greater proportion of cholesteryl esters compared to triglyceride in the core, because of the stimulated transport of CE from the expanded pool in the liver.(ABSTRACT TRUNCATED AT 400 WORDS)     

Altered glutathione homeostasis in heart augments cardiac lipotoxicity associated with diet-induced obesity in mice

Obesity-related cardiac lipid accumulation is associated with increased myocardial oxidative stress. The role of the antioxidant glutathione in cardiac lipotoxicity is unclear. Cystathionine β-synthase (Cbs) catalyzes the first step in the trans-sulfuration of homocysteine to cysteine, which is estimated to provide ～50% of cysteine for hepatic glutathione biosynthesis. As cardiac glutathione is a reflection of the liver glutathione pool, we hypothesize that mice heterozygous for targeted disruption of Cbs (Cbs(+/-)) are more susceptible to obesity-related cardiolipotoxicity because of impaired liver glutathione synthesis. Cbs(+/+) and Cbs(+/-) mice were fed a high fat diet (60% energy) from weaning for 13 weeks to induce obesity and had similar increases in body weight and body fat. This was accompanied by increased hepatic triglyceride but no differences in hepatic glutathione levels compared with mice fed chow. However, Cbs(+/-) mice with diet-induced obesity had greater glucose intolerance and lower total and reduced glutathione levels in the heart, accompanied by lower plasma cysteine levels compared with Cbs(+/+) mice. Higher triglyceride concentrations, increased oxidative stress, and increased markers of apoptosis were also observed in heart from Cbs(+/-) mice with diet-induced obesity compared with Cbs(+/+) mice. This study suggests a novel role for Cbs in maintaining the cardiac glutathione pool and protecting against cardiac lipid accumulation and oxidative stress during diet-induced obesity in mice.     

PPARalpha deficiency increases secretion and serum levels of apolipoprotein B-containing lipoproteins.

This study investigates the importance of peroxisome proliferator activated receptor alpha (PPARalpha) for serum apolipoprotein B (apoB) levels and hepatic secretion of apoB-containing lipoproteins. Total serum apoB and VLDL-apoB levels were higher in female PPARalpha-null mice compared with female wild-type mice, but no difference was seen in male mice. Furthermore, hepatic triglyceride secretion rate, determined in vivo after Triton WR1339 injection, was 2.4-fold higher in female PPARalpha-null mice compared with female wild-type mice, but no difference was observed in male mice. However, when fed a high fat diet, male PPARalpha-null mice displayed 2-fold higher serum levels of apoB and LDL cholesterol compared with male wild-type mice, but triglyceride levels were not affected. Hepatic LDL receptor protein levels were not influenced by PPARalpha deficiency, gender, or the fat diet. Hepatocyte cultures from female PPARalpha-null mice (cultured for 4 days in serum free medium) showed 2-fold higher total apoB secretion and increased secretion of apoB-48 VLDL, as well as 2.7-fold larger accumulation of VLDL-triglycerides in the medium compared with wild-type cultures. In conclusion, PPARalpha-deficient female mice, but not males, display high serum apoB associated with VLDL and increased hepatic triglyceride secretion. Moreover, male PPARalpha-null mice show increased susceptibility to high fat diet in terms of serum apoB levels.     

Effects of dietary fats and cholesterol on liver lipid content and hepatic apolipoprotein A-I, B, and E and LDL receptor mRNA levels in cebus monkeys.

The effects of the long-term administration of the dietary fats coconut oil and corn oil at 31% of calories with or without 0.1% (wt/wt) dietary cholesterol on plasma lipoproteins, apolipoproteins (apo), hepatic lipid content, and hepatic apoA-I, apoB, apoE, and low density lipoprotein (LDL) receptor mRNA abundance were examined in 27 cebus monkeys. Relative to the corn oil-fed animals, no significant differences were noted in any of the parameters of the corn oil plus cholesterol-fed group. In animals fed coconut oil without cholesterol, significantly higher (P less than 0.05) plasma total cholesterol (145%), very low density lipoprotein (VLDL) + LDL (201%) and high density lipoprotein (HDL) (123%) cholesterol, apoA-I (103%), apoB (61%), and liver cholesteryl ester (263%) and triglyceride (325%) levels were noted, with no significant differences in mRNA levels relative to the corn oil only group. In animals fed coconut oil plus cholesterol, all plasma parameters were significantly higher (P less than 0.05), as were hepatic triglyceride (563%) and liver apoA-I (123%) and apoB (87%) mRNA levels relative to the corn oil only group, while hepatic LDL receptor mRNA (-29%) levels were significantly lower (P less than 0.05). Correlation coefficient analyses performed on pooled data demonstrated that liver triglyceride content was positively associated (P less than 0.05) with liver apoA-I and apoB mRNA levels and negatively associated (P less than 0.01) with hepatic LDL receptor mRNA levels. Liver free and esterified cholesterol levels were positively correlated (P less than 0.05) with liver apoE mRNA levels and negatively correlated (P less than 0.025) with liver LDL receptor mRNA levels. Interestingly, while a significant correlation (P less than 0.01) was noted between hepatic apoA-I mRNA abundance and plasma apoA-I levels, no such relationship was observed between liver apoB mRNA and plasma apoB levels, suggesting that the hepatic mRNA of apoA-I, but not that of apoB, is a major determinant of the circulating levels of the respective apolipoprotein. Our data indicate that a diet high in saturated fat and cholesterol may increase the accumulation of triglyceride and cholesterol in the liver, each resulting in the suppression of hepatic LDL receptor mRNA levels. We hypothesize that such elevations in hepatic lipid content differentially alter hepatic apoprotein mRNA levels, with triglyceride increasing hepatic mRNA concentrations for apoA-I and B and cholesterol elevating hepatic apoE mRNA abundance.     

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.     

Comparison of the pharmacological profiles of murine antisense oligonucleotides targeting apolipoprotein B and microsomal triglyceride transfer protein

Therapeutic agents that suppress apolipoprotein B (apoB) and microsomal triglyceride transfer protein (MTP) levels/activity are being developed in the clinic to benefit patients who are unable to reach target LDL-C levels with maximally tolerated lipid-lowering drugs. To compare and contrast the metabolic consequences of reducing these targets, murine-specific apoB or MTP antisense oligonucleotides (ASOs) were administered to chow-fed and high fat-fed C57BL/6 or to chow-fed and Western diet-fed LDLr^−/− mice for periods ranging from 2 to 12 weeks, and detailed analyses of various factors affecting fatty acid metabolism were performed. Administration of these drugs significantly reduced target hepatic mRNA and protein, leading to similar reductions in hepatic VLDL/triglyceride secretion. MTP ASO treatment consistently led to increases in hepatic triglyceride accumulation and biomarkers of hepatotoxicity relative to apoB ASO due in part to enhanced expression of peroxisome proliferator activated receptor γ target genes and the inability to reduce hepatic fatty acid synthesis. Thus, although both drugs effectively lowered LDL-C levels in mice, the apoB ASO produced a more positive liver safety profile.     

CP-346086: an MTP inhibitor that lowers plasma cholesterol and triglycerides in experimental animals and in humans

A microsomal triglyceride transfer protein (MTP) inhibitor, CP-346086, was identified that inhibited both human and rodent MTP activity [concentration giving half-maximal inhibition (IC50) 2.0 nM]. In Hep-G2 cells, CP-346086 inhibited apolipoprotein B (apoB) and triglyceride secretion (IC50 2.6 nM) without affecting apoA-I secretion or lipid synthesis. When administered orally to rats or mice, CP-346086 lowered plasma triglycerides [dose giving 30% triglyceride lowering (ED30) 1.3 mg/kg] 2 h after a single dose. Coadministration with Tyloxapol demonstrated that triglyceride lowering was due to inhibition of hepatic and intestinal triglyceride secretion. A 2 week treatment with CP-346086 lowered total, VLDL, and LDL cholesterol and triglycerides dose dependently with 23%, 33%, 75%, and 62% reductions at 10 mg/kg/day. In these animals, MTP inhibition resulted in increased liver and intestinal triglycerides when CP-346086 was administered with food. When dosed away from meals, however, only hepatic triglycerides were increased. When administered as a single oral dose to healthy human volunteers, CP-346086 reduced plasma triglycerides and VLDL cholesterol dose dependently with ED50s of 10 mg and 3 mg, and maximal inhibition (100 mg) of 66% and 87% when measured 4 h after treatment. After a 2 week treatment (30 mg/day), CP-346086 reduced total and LDL cholesterol and triglycerides by 47%, 72%, and 75%, relative to either individual baselines or placebo, with little change in HDL cholesterol. Together, these data support further evaluation of CP-346086 in hyperlipidemia.     

Fish oil decreases hepatic cholesteryl ester secretion but not apoB secretion in African green monkeys

Two groups of African green monkeys were fed diets containing 40% of calories as fat with half of the fat calories as either fish oil or lard. The fish oil-fed animals had lower cholesterol concentrations in blood plasma (33%) and low density lipoproteins (LDL) (34%) than did animals fed lard. Size and cholesteryl ester (CE) content of LDL, strong predictors of coronary artery atherosclerosis in monkeys, were significantly less for the fish oil-fed animals although the apoB and LDL particle concentrations in plasma were similar for both diet groups. We hypothesized that decreased hepatic CE secretion led to the smaller size and reduced CE content of LDL in the fish oil-fed animals. Hepatic CE secretion was studied using recirculating perfusion of monkey livers that were infused during perfusion with fatty acids (85% 18:1 and 15% n-3) at a rate of 0.1 mumol/min per g liver. The rate of cholesterol secretion was less (P = 0.055) for the livers of fish oil versus lard-fed animals (3.3 +/- 0.5 vs. 6.0 +/- 1.2 mg/h per 100 g, mean +/- SEM) but the rate of apoB secretion was similar for both groups (0.92 +/- 0.15 vs. 1.01 +/- 0.13 mg/h per 100 g, respectively). The hepatic triglyceride secretion rate was also less (P less than 0.05) for the fish oil-fed animals (8.3 +/- 2.5 vs. 18.3 +/- 4.4 mg/h per 100 g). Liver CE content was lower (P less than 0.006) in fish oil-fed animals (4.1 +/- 0.8 vs. 7.4 +/- 0.7 mg/g) and this was reflected in a lower (P less than 0.04) esterified to total cholesterol ratio of perfusate VLDL (0.21 +/- 0.045 vs. 0.41 +/- 0.06). The hepatic VLDL of animals fed fish oil had 40-50% lower ratios of triglyceride to protein and total cholesterol to protein. From these data we conclude that livers from monkeys fed fish oil secreted similar numbers of VLDL particles as those of lard-fed animals although the hepatic VLDL of fish oil-fed animals were smaller in size and relatively enriched in surface material and depleted of core constituents. Positive correlations between plasma LDL size and both hepatic CE content (r = 0.87) and hepatic VLDL cholesterol secretion rate (r = 0.84) were also found.(ABSTRACT TRUNCATED AT 400 WORDS)     

The G0/G1 Switch Gene 2 Is an Important Regulator of Hepatic Triglyceride Metabolism

Nonalcoholic fatty liver disease is associated with obesity and insulin resistance. Factors that regulate the disposal of hepatic triglycerides contribute to the development of hepatic steatosis. G₀/G₁ switch gene 2 (G0S2) is a target of peroxisome proliferator-activated receptors and plays an important role in regulating lipolysis in adipocytes. Therefore, we investigated whether G0S2 plays a role in hepatic lipid metabolism. Adenovirus-mediated expression of G0S2 (Ad-G0S2) potently induced fatty liver in mice. The liver mass of Ad-G0S2-infected mice was markedly increased with excess triglyceride content compared to the control mice. G0S2 did not change cellular cholesterol levels in hepatocytes. G0S2 was found to be co-localized with adipose triglyceride lipase at the surface of lipid droplets. Hepatic G0S2 overexpression resulted in an increase in plasma Low-density lipoprotein (LDL)/Very-Low-density (VLDL) lipoprotein cholesterol level. Plasma High-density lipoprotein (HDL) cholesterol and ketone body levels were slightly decreased in Ad-G0S2 injected mice. G0S2 also increased the accumulation of neutral lipids in cultured HepG2 and L02 cells. However, G0S2 overexpression in the liver significantly improved glucose tolerance in mice. Livers expressing G0S2 exhibited increased 6-(N-(7-nitrobenz-2-oxa-1-3-diazol-4-yl) amino)-6-deoxyglucose uptake compared with livers transfected with control adenovirus. Taken together, our results provide evidence supporting an important role for G0S2 as a regulator of triglyceride content in the liver and suggest that G0S2 may be a molecular target for the treatment of insulin resistance and other obesity-related metabolic disorders.     

Feeding the nitric oxide synthase inhibitor L-N(omega)nitroarginine elevates serum very low density lipoprotein and hepatic triglyceride synthesis in rats

This study was conducted to study the influence of dietary L-N(omega)nitroarginine (L-NNA), a nitric oxide (NO) synthase inhibitor, on serum lipids and lipoproteins and on the activities of enzymes related to lipid metabolism in rats. Feeding rats a diet containing 0.2 g/kg L-NNA for 5 weeks elevated serum concentrations of triglyceride, cholesterol, phospholipid, and free fatty acid and reduced serum nitrate (an oxidation product of NO). The elevation in serum triglyceride was mainly due to the elevation in very low density lipoprotein (VLDL) triglyceride. Contents of cholesterol and phospholipid in the VLDL fraction also were elevated by L-NNA. L-NNA treatment caused significantly higher activity of hepatic microsomal phosphatidate phosphohydrolase (the rate-limiting enzyme in triglyceride synthesis) and lower activity of hepatic carnitine palmitoyltransferase (the rate-limiting enzyme in fatty acid oxidation). Activities of hepatic enzymes responsible for fatty acid synthesis such as glucose-6-phosphate dehydrogenase, malic enzyme, and fatty acid synthase were unaffected by L-NNA. The activity of hepatic microsomal phosphocholine cytidyltransferase (the rate-limiting enzyme in phosphatidylcholine synthesis) was reduced significantly by L-NNA. Our results suggest that lower NO production caused the elevations in hepatic triglyceride synthesis by higher esterification of fatty acid and lower fatty acid oxidation, leading to an enrichment of VLDL triglyceride.     

Hepatic overexpression of microsomal triglyceride transfer protein (MTP) results in increased in vivo secretion of VLDL triglycerides and apolipoprotein B.

The microsomal triglyceride transfer protein (MTP) is essential for the hepatic secretion of apolipoprotein (apo) B-containing lipoproteins. Previous studies have indicated that inhibition of MTP results in decreased apoB plasma levels and decreased hepatic triglyceride secretion. However, the metabolic effects of overexpression of MTP have not been investigated. We constructed a recombinant adenovirus expressing MTP (AdhMTP) and used it to assess the effects of hepatic overexpression of MTP in mice. Injection of AdhMTP into C57BL/6 mice resulted in a 3-fold increase in hepatic microsomal triglyceride transfer activity compared to mice injected with Adnull. On day 4 after virus injection, AdhMTP-injected mice had significantly elevated plasma TG levels as compared to control virus (Adnull)-injected mice. Hepatic TG secretion rates were significantly greater in AdhMTP-injected mice (184 +/- 12 mg/kg/h) compared with Adnull-injected mice (65 +/- 9 mg/kg/h, P < 0.001). In addition, hepatic very low density lipoprotein (VLDL) apoB secretion in the AdhMTP-injected group was 74% higher than in the control virus group. Hepatic secretion of apoB-48 and apoB-100 contributed equally to this increase.These results provide the first data that hepatic overexpression of MTP results in increased secretion of VLDL-triglycerides as well as VLDL-apoB in vivo. These results suggest that MTP is rate-limiting for VLDL apoB secretion in wild-type mice under basal chow-fed conditions.