Apolipoprotein Gene Expression Research Articles

Circadian Regulation of Apolipoproteins in the Brain: Implications in Lipid Metabolism and Disease.

The circadian rhythm is a 24 h internal clock within the body that regulates various factors, including sleep, body temperature, and hormone secretion. Circadian rhythm disruption is an important risk factor for many diseases including neurodegenerative illnesses. The central and peripheral oscillators' circadian clock network controls the circadian rhythm in mammals. The clock genes govern the central clock in the suprachiasmatic nucleus (SCN) of the brain. One function of the circadian clock is regulating lipid metabolism. However, investigations of the circadian regulation of lipid metabolism-associated apolipoprotein genes in the brain are lacking. This review summarizes the rhythmic expression of clock genes and lipid metabolism-associated apolipoprotein genes within the SCN in Mus musculus. Nine of the twenty apolipoprotein genes identified from searching the published database (SCNseq and CircaDB) are highly expressed in the SCN. Most apolipoprotein genes (ApoE, ApoC1, apoA1, ApoH, ApoM, and Cln) show rhythmic expression in the brain in mice and thus might be regulated by the master clock. Therefore, this review summarizes studies on lipid-associated apolipoprotein genes in the SCN and other brain locations, to understand how apolipoproteins associated with perturbed cerebral lipid metabolism cause multiple brain diseases and disorders. This review describes recent advancements in research, explores current questions, and identifies directions for future research.

Hypercholesterolemia Induced by Cholesterol- or Cystine-Enriched Diets is Characterized by Different Plasma Lipoprotein and Apolipoprotein Concentrations in Rats

This study examined the effects of diet-induced hypercholesterolemia on plasma apolipoprotein (apo) concentrations and hepatic apolipoprotein mRNA levels in rats. Hypercholesterolemia was induced by feeding rats diets containing an excess of either cholesterol or cystine. After cholesterol feeding, plasma apo E and apo B concentrations were lower (-65%, P < 0.001) and greater (+39%, P < 0.01), respectively, compared with control diet-fed rats. After cystine feeding, plasma apo B and apo E concentrations were greater (+46%, P < 0.01 and +75%, P < 0.001, respectively) and plasma apo A-IV concentration was lower (-29%, P < 0.001) than in rats fed control diet. After cholesterol or cystine feeding, a tendency (one-way ANOVA, P = 0.08) for greater apo B mRNA level (+42% and +47%, respectively) was observed compared with control diet-fed rats. No difference emerged between groups for apo E and apo A-I mRNA levels. An opposite effect of cholesterol and cystine feeding was shown for apo A-IV mRNA level, i.e., higher after cholesterol feeding (+47%, P < 0.05) and lower after cystine feeding (-65%, P < 0.01). From this work, it seems that hypercholesterolemia induced by dietary cholesterol or by increased cholesterogenesis in cystine-fed rats is characterized by different plasma lipoprotein and apolipoprotein concentrations and is associated with different apolipoprotein gene expression in the liver.

Effects of cottonseed meal on performance, gossypol residue, liver function, lipid metabolism, and cecal microbiota in geese.

A total of 240 28-d-old male goslings were used to investigate the effects of cottonseed meal (CSM) on performance, gossypol residue, liver function, lipid metabolism, and cecal microbiota. All birds were randomly allotted into five groups (eight goslings/replicate, six replicates/group) and subjected to a 35-d experiment. Five isonitrogenous and isoenergetic diets were formulated to produce diets in which 0% (control), 25% (CSM25), 50% (CSM50), 75% (CSM75), and 100% (CSM100) of protein from soybean meal was replaced by protein from CSM. The free gossypol contents in the five diets were 0, 44, 92, 135, and 183 mg/kg, respectively. Dietary CSM did not affect the growth performance from 29 to 63 d and carcass traits at 63 d (P > 0.05). Liver gossypol residues were influenced (P < 0.05) by dietary CSM and increased linearly (P < 0.05) and quadratically (P < 0.05) as dietary CSM increased. The malondialdehyde content of the liver was lower in the CSM100 group than in the other groups (P < 0.05). Serum triglyceride and low-density lipoprotein cholesterol were influenced (P < 0.05) by dietary CSM and increased linearly (P < 0.05) with increasing dietary CSM. Dietary CSM altered (P < 0.05) the composition of some fatty acids in the liver and breast muscle. The concentration of linolenic acid and Σn-3 polyunsaturated fatty acid (PUFA) in the liver and breast muscle decreased linearly, but the Σn-6/Σn-3 PUFA ratio increased linearly with increasing dietary CSM (P < 0.05). Dietary CSM affected (P < 0.05) the hepatic gene expression of fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), and apolipoprotein B (ApoB). As the dietary CSM concentration increased, the hepatic gene expression of FAS increased linearly (P < 0.05) and quadratically (P < 0.05), but the hepatic gene expression of ACC and ApoB increased linearly (P < 0.05). The CSM diet decreased the relative abundance of the Bacteroidota and Bacteroides (P < 0.05), and the CSM50 diet increased the relative abundance of the Firmicutes and Colidextribacter (P < 0.05) compared to the control group. Overall, these results show that dietary CSM has no adverse effects on the performance of goslings from 29 to 63 d. However, CSM affected organismal lipid metabolism, reduced products' edible value, and adaptively altered cecum microbiota.

The toxic effects of chronic atrazine exposure on the intestinal microbiota, metabolism and transcriptome of Pelophylax nigromaculatus larvae

Atrazine, a widely used herbicide, is a potential threat to the intestines of animals due to its long-term persistence in water and accumulation in animal tissues, but the underlying mechanism is unclear. In this study, we evaluated the toxicity of atrazine (50, 100, and 500 µg/L) on the intestine of Pelophylax nigromaculatus larvae from Gosner stages 18–42 (about 90 days). We conducted a 16S rRNA gene amplification study, which showed that chronic exposure to atrazine changed the diversity and composition of intestinal microbiota of P. nigromaculatus larvae. Chronic exposure to atrazine (50, 100, and 500 µg/L) significantly decreased the relative abundance of Fusobacteria and U114, while 50 µg/L and 100 µg/L atrazine resulted in a significant increase of Clavibacter. The LC-MS/MS metabolomics indicated that there were 145 significantly changed metabolites. Furthermore, atrazine led to metabolic abnormalities related to purine metabolism and biosynthesis of amino acids in the intestine. The transcriptomic results showed that atrazine exposure caused 21,120 differentially expressed genes between the control and the atrazine exposure groups. Atrazine exposure primarily disturbed the pathways related to the cytokine-cytokine receptor interaction and cell adhesion molecules. Moreover, 500 µg/L atrazine caused a significant up-regulation in the gene expression of apolipoprotein (ApoA1, ApoB, and ApoA4), and fatty acid binding protein (FABP1), as well as a significant down-regulation in the gene expression of superoxide dismutase (SOD). Our work provides insight into the ecotoxicity mechanisms of atrazine in amphibians at the level of intestinal microbiota response, metabolome and transcriptome, and serves as a basis for future research on pesticide pollutants.

The Roles of Fatty Acids and Apolipoproteins in the Kidneys.

The kidneys are organs that require energy from the metabolism of fatty acids and glucose; several studies have shown that the kidneys are metabolically active tissues with an estimated energy requirement similar to that of the heart. The kidneys may regulate the normal and pathological function of circulating lipids in the body, and their glomerular filtration barrier prevents large molecules or large lipoprotein particles from being filtered into pre-urine. Given the permeable nature of the kidneys, renal lipid metabolism plays an important role in affecting the rest of the body and the kidneys. Lipid metabolism in the kidneys is important because of the exchange of free fatty acids and apolipoproteins from the peripheral circulation. Apolipoproteins have important roles in the transport and metabolism of lipids within the glomeruli and renal tubules. Indeed, evidence indicates that apolipoproteins have multiple functions in regulating lipid import, transport, synthesis, storage, oxidation and export, and they are important for normal physiological function. Apolipoproteins are also risk factors for several renal diseases; for example, apolipoprotein L polymorphisms induce kidney diseases. Furthermore, renal apolipoprotein gene expression is substantially regulated under various physiological and disease conditions. This review is aimed at describing recent clinical and basic studies on the major roles and functions of apolipoproteins in the kidneys.

Circadian regulation of apolipoprotein gene expression affects testosterone production in mouse testis

The circadian clock system plays an important role in regulating testosterone synthesis in mammals. Male Bmal1−/− mice are infertile with low serum testosterone levels and decreased expression of testicular steroidogenic genes, suggesting that circadian clock genes regulate testosterone biosynthesis by activating steroidogenic gene transcription. However, whether the circadian clock regulates testosterone production via other genes remains unknown. Using Bmal1−/− mice and their wild-type (WT) siblings, we aimed to identify additional genes by which the circadian clock regulates testosterone synthesis. WT and Bmal1−/− mouse testes sections had similar normal morphologies, although there was a decrease in testicular spermatozoa in the Bmal1−/− mice. Low serum testosterone levels were detected in the Bmal1−/− mice. RNA sequencing identified 37 and 48 genes that were differentially expressed between WT and Bmal1−/− mouse testes at circadian time (CT2 and CT14), respectively. The cholesterol metabolism pathway was significantly enriched in the KEGG pathway analysis, and there was lower expression of three apolipoprotein genes (Apoa1, Apoa2, and Apoc3) at CT2 in the testes of Bmal1−/− mice than in those of WT mice. These decreases in Apoa1, Apoa2, and Apoc3 expression were verified by quantitative polymerase chain reaction analysis, which also revealed downregulation of the expression of the circadian clock (Per2, Dbp, and Nr1d1) and steroidogenic (StAR, Cyp11a1, and Hsd17b3) genes. The expression of circadian clock genes was relatively stable in WT mice over a 20-h period, whereas there was clear circadian rhythmic expression of Apoa1, Apoa2, Apoc3, StAR, Cyp11a1, Hsd3b2, and Hsd17b3. Bmal1−/− mice showed severely reduced expression of testicular circadian clock genes at three time points (CT4, CT12, and CT20), and a reduction in mRNA expression levels of Apo (Apoa1, Apoa2, and Apoc3) and steroidogenic (StAR, Cyp11a1, Hsd3b2, and Hsd17b3) genes. Oil Red O staining showed decreased lipid aggregation in the Leydig cells of Bmal1−/− mouse testes. Considering the vital role of Apo genes in high-density lipoprotein formation and cholesterol transport, the present data suggest that the circadian clock system regulates testosterone production by orchestrating the rhythmic expression of Apo genes. These data extend our understanding of the role of the circadian clock in regulating testosterone production in mammals.

A Model Construction of Starvation Induces Hepatic Steatosis and Transcriptome Analysis in Zebrafish Larvae.

Simple SummaryNonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disorder. Previous studies have focused on NAFLD caused by factors such as obesity or high-fat diets, but in recent years, more and more studies have proved that starvation is also an important cause of NAFLD. It has shown great harm in human health (e.g., dieting and anorexia nervosa) and farming economic benefits (e.g., laying hens and mink). However, the molecular mechanism underlying of starvation-induced NAFLD remain unclear. Regarding the study of NAFLD, zebrafish is currently widely used model organism. Thus, in this study, we used zebrafish to establish a starvation-induced hepatic steatosis model. As we all know, hepatic steatosis is usually a prerequisite for NAFLD. Subsequently, we performed a comparative transcriptome analysis of starvation-induced hepatic steatosis by RNA-Seq. We demonstrated that starvation triggers hepatic steatosis by promoting extrahepatic fatty acid uptake and lipogenesis, and inhibits hepatic fatty acid metabolism and lipid transport. Based on the indications provided by these data, we further revealed that cd36 plays a crucial role in regulating extrahepatic fatty acid uptake and inducing hepatic steatosis during starvation conditions. Altogether, these findings will help us understand the pathogenesis of starvation-induced NAFLD.Hepatic steatosis caused by starvation, resulting in non-alcoholic fatty liver disease (NAFLD), has been a research topic of human clinical and animal experiments. To understand the molecular mechanisms underlying the triggering of abnormal liver metabolism by starvation, thus inducing hepatic lipid accumulation, we used zebrafish larvae to establish a starvation-induced hepatic steatosis model and conducted comparative transcriptome analysis by RNA-seq. We demonstrated that the incidence of larvae steatosis is positively correlated with starvation time. Under starvation conditions, the fatty acid transporter (slc27a2a and slc27a6-like) and fatty acid translocase (cd36) were up-regulated significantly to promote extrahepatic fatty acid uptake. Meanwhile, starvation inhibits the hepatic fatty acid metabolism pathway but activates the de novo lipogenesis pathway to a certain extent. More importantly, we detected that the expression of numerous apolipoprotein genes was downregulated and the secretion of very low density lipoprotein (VLDL) was inhibited significantly. These data suggest that starvation induces hepatic steatosis by promoting extrahepatic fatty acid uptake and lipogenesis, and inhibits hepatic fatty acid metabolism and lipid transport. Furthermore, we found that starvation-induced hepatic steatosis in zebrafish larvae can be rescued by targeting the knockout cd36 gene. In summary, these findings will help us understand the pathogenesis of starvation-induced NAFLD and provide important theoretical evidence that cd36 could serve as a potential target for the treatment of NAFLD.

Dietary supplementation of bile acid attenuate adverse effects of high-fat diet on growth performance, antioxidant ability, lipid accumulation and intestinal health in juvenile largemouth bass (Micropterus salmoides)

Bile acid (BA) has been reported to improve growth performance and play an important role in lipid metabolism of fish. Five diets were formulated to investigate the effects of dietary bile acid (chenodeoxycholic acid) supplementation on growth performance, antioxidant capacity, lipid metabolism and intestinal health of juvenile largemouth bass (Micropterus salmoides). Fish (18.35 ± 0.05 g) were randomly fed five diets: the positive control diet (10.87% lipid, C), high fat diet (18.08% lipid, HF), and HF diets supplemented with 300, 600 and 900 mg/kg chenodeoxycholic acid (HFC1, HFC2 and HFC3, respectively). After 9 weeks of feeding experiment, the weight gain (WG) and special growth rate (SGR) were significantly lower in the fish fed diet HF compared with those fed diet C (P < 0.05). Compared with the group C, there were no significant differences in WG and SGR of fish fed high fat diet with 900 mg/kg chenodeoxycholic acid (P > 0.05). Viscerosomatic index (VSI) and intraperitoneal fat ratio (IPF) were significantly higher in fish fed high fat diets than those fed the control diet (P < 0.05). The crude lipid and protein contents in muscle showed no significant differences among fish fed diets with high dietary lipid (P > 0.05), while the crude lipid of muscle significantly decreased in HFC2 group (P < 0.05). Triglyceride (TG), malondialdehyde (MDA) in plasma and MDA in liver were higher in HF group than control group, addition of BA could decrease those contents effectively and increase the gene expression of SOD and GSH-Px. Lipidprotein transport and cholesterol synthesis related mRNA levels were significantly increased in fish fed high fat diets containing BA. The supplementation of 900 mg/kg BA significantly increased the gene expression of apolipoprotein B (ApoB-100), sterol 26-hydroxylase (CYP27α) and liver X receptor (LXR) (P < 0.05). High-fat diet significantly increased the pro-apoptotic gene expression, while anti-apoptotic gene B-cell lymphoma-2 (Bcl-2) and B-cell lymphoma-xl (Bcl-xl) mRNA levels were significantly increased with the increasing dietary BA (P < 0.05). In addition, supplemental BA in high-fat diet increased the height of intestinal fold and decreased lipid accumulation in liver. These results suggested that dietary BA supplementation could improve the digestion and absorption of lipids, antioxidant capacity and intestinal health, thereby attenuating the adverse effect induced by high fat diet of largemouth bass.

Turnover of bile acids in liver, serum and caecal content by high-fat diet feeding affects hepatic steatosis in rats

BackgroundBile acids (BAs) participate in lipid absorption and serve as metabolic regulatory factors in gut-liver communication. To date, there are no studies on the systemic patterns of BAs in the serum, liver, and gut in the same non-alcoholic fatty liver disease (NAFLD) model. MethodsA targeted metabolomics approach and 16S rRNA sequencing were used to identify the profile of BAs and connection between BAs and microbiota. The role and mechanism of altered BAs on hepatic steatosis were investigated. FindingsIn the liver, the composition of taurocholic acid (TCA) was increased, but taurohyodeoxycholic acid (THDCA) and ursodeoxycholic acid (UDCA) were decreased. In the gut, the deconjugated form of TCA (cholic acid (CA)) was increased, while the deconjugated forms of THDCA (α-hyodeoxycholic acid (HDCA)) and ω-muricholic acid (ωMCA) were decreased. In the serum, the composition of TCA was increased, while both HDCA and THDCA were decreased. THDCA induced the gene expression of apolipoprotein, bile secretion-related proteins, and cytochrome P450 family but suppressed inflammatory response genes expression in steatotic hepatocytes by RNAseq analysis. THDCA ameliorated neutral lipid accumulation and improved insulin sensitivity in primary rat hepatocytes. The decreased HDCA level correlated with the level of Bacteroidetes, while the level of CA correlated with the levels of Firmicutes and Verrucomicrobia but correlated inversely with Bacteroidetes. ConclusionBAs profiles in the serum, liver and caecal content were altered in a rat NAFLD model, which may affect hepatic lipid accumulation and correlate with gut dysbiosis.

Metabolism‐dependent Induction by Fructose of Lipogenic Gene Expression Occurs in Mouse Enterocytes and not in other Cell Types

High intakes of fructose are associated with hypertriglyceridemia attributed primarily to hepatic conversion of fructose to fats. Even though the small intestine expresses lipogenic genes, is luminally exposed to high fructose, and can convert labelled fructose to fatty acids, there is little information on the effect of fructose on expression of small intestinal lipogenic genes, and on the identity of intestinal cell type(s) capable of fructose‐induced regulation. We hypothesized that fructose regulates the intestinal expression of genes involved in lipid and chylomicron synthesis and that regulation depends on the fructose transporter GLUT5 and on the enzyme ketohexokinase (KHK). Because intestinal stem cells can differentiate into absorptive enterocytes and secretory goblet, Paneth and other cells, we next hypothesized that fructose regulation of lipogenic genes is specific to enterocytes and not to other cell types. We fed for 3 d fructose or glucose solutions by gavage to wildtype, GLUT5‐KO and KHK‐KO mice with free access to food, then expression of representative genes involved in lipid and chylomicron synthesis was determined. We then isolated stem cells from chow‐fed wildtype, GLUT5‐KO and KHK‐KO mice and directed the differentiation of these cells into organoids highly enriched in enterocytes (90%), goblet (80%), Paneth (65%) and stem (90%) cells, incubated them in 10 mM fructose or glucose solutions, then determined mRNA levels of lipogenic genes. In vivo mRNA and protein expression of representative genes involved in lipid synthesis (sterol responsive element binding protein‐1c, acetyl‐CoA carboxylase 1, fatty acid synthase, stearoyl‐CoA desaturase 1, diacylglycerol acyltransferase 1 and glycerol kinase) and intestine‐specific apolipoproteins APO‐A4 and APO‐B48 each increased markedly in wildtype, but not in GLUT5‐KO and KHK‐KO, mice fed fructose. In contrast, expression of the nonfructolytic nonlipogenic glucose transporter SGLT1 was independent of diet. GLUT5‐ and KHK‐dependent, fructose‐induced increases in mRNA levels of these genes were observed only in enterocytes in vitro. Dietary fructose specifically stimulates the expression of lipogenic and apolipoprotein genes in the small intestine, and these effects require fructose transport and metabolism. Thus, ingested fructose must be absorbed by GLUT5 and not merely “tasted” by numerous sweet receptors expressed in the gut, then phosphorylated by KHK to fructose‐1‐phosphate. Moreover, fructose effects occur only in absorptive enterocytes, suggesting that the capacity for fructose modulation of these genes is lost during differentiation into secretory cell types.Support or Funding InformationSupported by NSF Grant IOS‐1456673 (RPF) and by NIH Grant R01‐DK‐102934 (NG).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Turnover of Bile Acids in Liver, Serum and Caecal Content by High-Fat Diet Feeding Affects Hepatic Steatosis in Rats

Abstract Background Bile acids (BAs) participate in lipid absorption and serve as metabolic regulatory factors in gut-liver communication. To date, there are no studies on the systemic patterns of BAs in the serum, liver, and gut in the same non-alcoholic fatty liver disease (NAFLD) model. Methods A targeted metabolomics approach and 16S rRNA sequencing were used to identify the profile of BAs and connection between BAs and microbiota. The role and mechanism of altered BAs on hepatic steatosis were investigated. Findings In the liver, the composition of taurocholic acid (TCA) was increased, but taurohyodeoxycholic acid (THDCA) and ursodeoxycholic acid (UDCA) were decreased. In the gut, the deconjugated form of TCA (cholic acid (CA)) was increased, while the deconjugated forms of THDCA (α-hyodeoxycholic acid (HDCA)) and ω-muricholic acid (ωMCA) were decreased. In the serum, the composition of TCA was increased, while both HDCA and THDCA were decreased. THDCA induced the gene expression of apolipoprotein, bile secretion-related proteins, and cytochrome P450 family but suppressed inflammatory response genes expression in steatotic hepatocytes by RNAseq analysis. THDCA ameliorated neutral lipid accumulation and improved insulin sensitivity in primary rat hepatocytes. The decreased HDCA level correlated with the level of Bacteroidetes, while the level of CA correlated with the levels of Firmicutes and Verrucomicrobia but correlated inversely with Bacteroidetes. Conclusion BAs profiles in the serum, liver and caecal content were altered in a rat NAFLD model, which may affect hepatic lipid accumulation and correlate with gut dysbiosis.

Evaluation of a synthetic emulsifier product supplementation on broiler chicks

The digestive physiology of young chickens is characterized by inefficient digestion and absorption of fat due to a low level of natural endogenous lipase production. These evidences have increased the interest on the use of emulsifiers to improve utilization of fats in young chickens and growth performance of broiler. The effect of a synthetic emulsifier on growth performance, meat quality, caecum microbial count, plasma metabolites and hepatic apolipoprotein gene expression was investigated in male broiler chicks. A total of 600 one-day-old ROSS 308 broiler chicks were assigned to 2 experimental groups consisting of 15 pens with 20 birds/ per pen each, to compare the different dietary treatments: control diet (CTR) or diet supplemented with AVI-MUL TOP (AMT) at 1 g/kg from day 0 to 12, 0.75 g/kg from day 12 to 22 and 0.5 g/kg from day 22 to 44. Growth performance was determined on days 0, 12, 22, and 44. At the end of the trial (day 44), one chick from each pen was chosen on body weight (BW) basis and sacrificed and samples of blood, liver, caecum content and breast were collected for analysis. AMT supplementation increased BW on days 12 and 22 (P = 0.02; P = 0.02) and ADG from day 0 to 12 (P = 0.02), while reduced FCR from day 22 to 44 (P = 0.047) and from day 0 to 44 (P = 0.02). AMT supplementation modified carcass and meat characteristics, increasing dressing percentage (P = 0.01) and b* (yellowness) (P = 0.01) compared to control group. Moreover, AMT dietary supplementation increased total cholesterol (P = 0.02) and HDL cholesterol (P = 0.02) plasma concentrations. No differences between the two treatments were observed in caecum microbial counts and hepatic apolipoprotein gene expression. In conclusion, our findings show that AMT supplementation to broiler chicks may have a beneficial effect on growth performances (BW, ADG and FCR) and carcass dressing and may affect meat colour (b* yellowness) and lipid metabolism (cholesterol and HDL).

Activation of liver X receptor suppresses angiogenesis via induction of ApoD.

Liver X receptors (LXRs) are important sensors and regulators for cholesterol, fatty acid, and glucose. LXRs play essential roles in the development and progression of cardiovascular diseases. We examined the effects of T0901317, a potent LXR agonist, on angiogenesis of human umbilical vein endothelial cells (HUVECs). Treatment with T0901317 inhibited the tube formation and migration of HUVECs and reduced the in vivo angiogenesis, as determined by chorioallantoic membrane assay. T0901317 stimulated gene and protein expression of LXR target gene apolipoprotein D (ApoD). Overexpression of ApoD suppressed the tube formation of HUVECs. ApoD interacted with scavenger receptor class B member 1 (SR-B1), while knockdown of SR-B1 blocked suppressive effects of T0901317 on HUVEC migration. T0901317 treatment or overexpression of ApoD lessened expression of proteins regulating angiogenesis, including phospho-eNOS S1177, phospho-Akt T308, phospho-Akt S473, eNOS, mammalian target of rapamycin, VEGF-A, VEGF-C, IL-8, RhoB, matrix metalloproteinase (MMP)-8, -9, and monocyte chemoattractant protein 1. Our study suggested that activation of LXR interferes with angiogenesis through induction of LXR target gene ApoD, which in turn suppresses PI3K-Akt-eNOS signaling, an essential pathway regulating angiogenesis. ApoD may be a potential therapeutic target for tumor angiogenesis.-Lai, C.-J., Cheng, H.-C., Lin, C.-Y., Huang, S.-H., Chen, T.-H., Chung, C.-J., Chang, C.-H., Wang, H.-D., Chuu, C.-P. Activation of liver X receptor suppresses angiogenesis via induction of ApoD.

Abstract 579: In vitro Models Concur With Clinical Results to Confirm Pleiotropic Mechanisms of Action for Gemcabene

Introduction: Gemcabene, a fraudulent fatty acid, induces biological properties in nonclinical models, of which many translate to clinical findings in dyslipidemia patients. Its known mechanism of action includes reduction of the overall hepatic de novo triglyceride and cholesterol synthesis, and increase in VLDL clearance. Clinical benefits in various human populations infer that other mechanisms are involved that warrant evaluation. Methods and Results: We assessed the gemcabene effects in other molecular mechanistic models, particularly to study inhibition of human ATP Citrate Lyase (hACL), apolipoprotein gene expression in Sandwich-Cultured Transporter Certified TM Human Hepatocytes (SCHH), and expression of genes related to inflammation and cell signaling. Gemcabene, MEDICA-16, palmitic acid and their CoA thioesters were studied in a recombinant hACL in vitro assay system. Gemcabene-CoA thioester, but not the parent compound, inhibited recombinant hACL activity in vitro in a dose-dependent manner, in agreement with results obtained with palmitoyl-CoA and palmitic acid. Unlike palmitic acid, MEDICA-16, and bempedoic acid, gemcabene does not form a CoA ester in human hepatocytes: radioisotope studies show 98.8% parent gemcabene. Further, gemcabene potential on the gene expression of lipogenesis and inflammation markers was assessed in SCHH. Gemcabene showed significant regulation of HMG-CoA synthase 2 (HMGCS2) and CRP mRNAs. A marked induction response of the HMGCS2 mRNA content was observed, ranging from 2.26- to 2.73-fold (p ≤ 0.05) over control in SCHH treated with all concentrations of gemcabene (500, 1000, and 1500 μM). Also, a clear and statistically significant (p-value ≤ 0.05) concentration-related suppression response of CRP mRNA content was observed, ranging from (-2.58)- to (-2.65)-fold below control in SCHH treated with 1000 and 1500 μM gemcabene. Finally, RT-PCR analysis of liver samples from STAM TM mice treated with gemcabene revealed its downregulating effect on inflammatory, lipogenic, lipoprotein metabolism, and cell signaling genes: TNF-α, MCP-1, NF-κB, ApoC-III, and ACC1. Conclusion: Gemcabene manifests its pharmacological profile in lipid management and inflammation by multiple mechanisms of action.

Active liver X receptor signaling in phagocytes in multiple sclerosis lesions

Objective: We sought to determine the liver X receptor (LXR) ligands present in human macrophages after myelin phagocytosis and whether LXRs are activated in multiple sclerosis (MS) lesions. Methods: We used real-time quantitative polymerase chain reaction (PCR) and immunohistochemistry to determine expression of LXRs and their response genes in human phagocytes after myelin phagocytosis and in active MS lesions. We used gas chromatographic/mass spectrometric analysis to determine LXR-activating oxysterols and cholesterol precursors present and formed in myelin and myelin-incubated cells, respectively. Results: Myelin induced LXR response genes ABCA1 and ABCG1 in human monocyte-derived macrophages. In active MS lesions, we found that both gene expression and protein levels of ABCA1 and apolipoprotein E (APOE) are upregulated in foamy phagocytes. Moreover, we found that the LXR ligand 27-hydroxycholesterol (27OHC) is significantly increased in human monocyte-derived macrophages after myelin uptake. Conclusion: LXR response genes are upregulated in phagocytes present in active MS lesions, indicating that LXRs are activated in actively demyelinating phagocytes. In addition, we have shown that myelin contains LXR ligands and that 27OHC is generated in human monocyte-derived macrophages after myelin processing. This suggests that LXRs in phagocytes in active MS lesions are activated at least partially by (oxy)sterols present in myelin and the generation thereof during myelin processing.

Milk Polar Lipids Affect In Vitro Digestive Lipolysis and Postprandial Lipid Metabolism in Mice1–3

Background: Polar lipid (PL) emulsifiers such as milk PLs (MPLs) may affect digestion and subsequent lipid metabolism, but focused studies on postprandial lipemia are lacking.Objective: We evaluated the impact of MPLs on postprandial lipemia in mice and on lipid digestion in vitro.Methods: Female Swiss mice were gavaged with 150 μL of an oil-in-water emulsion stabilized with 5.7 mg of either MPLs or soybean PLs (SPLs) and killed after 1, 2, or 4 h. Plasma lipids were quantified and in the small intestine, gene expression was analyzed by reverse transcriptase–quantitative polymerase chain reaction. Emulsions were lipolyzed in vitro using a static human digestion model; triglyceride (TG) disappearance was followed by thin-layer chromatography.Results: In mice, after 1 h, plasma TGs tended to be higher in the MPL group than in the SPL group (141 μg/mL vs. 90 μg/mL; P = 0.07) and nonesterified fatty acids (NEFAs) were significantly higher (64 μg/mL vs. 44 μg/mL; P < 0.05). The opposite was observed after 4 h with lower TGs (21 μg/mL vs. 35 μg/mL; P < 0.01) and NEFAs (20 μg/mL vs. 32 μg/mL; P < 0.01) in the MPL group compared with the SPL group. This was associated at 4 h with a lower gene expression of apolipoprotein B (Apob) and Secretion Associated, Ras related GTPase 1 gene homolog B (Sar1b), in the duodenum of MPL mice compared with SPL mice (P < 0.05). In vitro, during the intestinal phase, TGs were hydrolyzed more in the MPL emulsion than in the SPL emulsion (decremental AUCs were 1750%/min vs. 180%/min; P < 0.01). MPLs enhance lipid intestinal hydrolysis and promote more rapid intestinal lipid absorption and sharper kinetics of lipemia.Conclusions: Postprandial lipemia in mice can be modulated by emulsifying with MPLs compared with SPLs, partly through differences in chylomicron assembly, and TG hydrolysis rate as observed in vitro. MPLs may thereby contribute to the long-term regulation of lipid metabolism.

Hepatitis C virus infection alters lipid metabolism depending on IL28B polymorphism and viral genotype and modulates gene expression in vivo and in vitro

Hepatitis C virus (HCV) interacts with lipid receptors to enter the cell, circulates as lipoviroparticle and is secreted as VLDL. We aimed to investigate the role of the rs12979860 polymorphism in the IL28B gene in 143 with chronic hepatitis C genotype 1, 144 infected with genotype 3, 90 genotype 4 and 413 noninfected individuals on lipid profile and to test the impact of HCV infection in an in vitro model on VLDL biosynthesis-related gene expression rs12979860 polymorphism was analysed using real-time PCR coupled to Fluorescence Resonance Energy Transfer (FRET). Huh7.5 (rs12979860 CT) and Huh7 (genotype CC) cells were infected with JFH-1 particles and serum from patients infected with genotypes 1 and 3. Gene expression of apolipoprotein B (apoB), microsomal triglyceride transfer protein (MTP), acetyl CoA carboxylase (ACC), diacylglycerol acyltransferase 2 (DGAT2), diacylglycerol acyltransferase 1 (DGAT1) and low-density lipoprotein receptor (LDLr) genes were determined by semiquantitative RT-PCR in vivo and in vitro. Genotype CC rs12979860 polymorphism was associated with significantly higher serum LDL and total cholesterol levels in patients with hepatitis C genotype 1 but not in patients with hepatitis C genotype 3, genotype 4 and control (noninfected) population. Genotype CC was more often seen in genotype 3 and healthy people in comparison with genotype 1; P=0.001. In vitro results showed that HCV infection promotes lipid metabolism gene expression induction depending on viral genotype, but to a lesser extent in cells with CT genotype. These results demonstrate that IL28B genotype influences lipid metabolism in patients with hepatitis C but not in noninfected and it seems to be viral genotype-mediated. HCV infection modifies lipid-related genes expression (DGAT1 and DGAT2) in cultured cells based on viral genotype and IL28 polymorphism.

Exon Skipping of Hepatic APOB Pre-mRNA With Splice-switching Oligonucleotides Reduces LDL Cholesterol In Vivo

Familial hypercholesterolemia (FH) is a genetic disorder characterized by extremely high levels of plasma low-density lipoprotein (LDL), due to defective LDL receptor-apolipoprotein B (APOB) binding. Current therapies such as statins or LDL apheresis for homozygous FH are insufficiently efficacious at lowering LDL cholesterol or are expensive. Treatments that target APOB100, the structural protein of LDL particles, are potential therapies for FH. We have developed a series of APOB-directed splice-switching oligonucleotides (SSOs) that cause the expression of APOB87, a truncated isoform of APOB100. APOB87, like similarly truncated isoforms expressed in patients with a different condition, familial hypobetalipoproteinemia, lowers LDL cholesterol by inhibiting very low-density lipoprotein (VLDL) assembly and increasing LDL clearance. We demonstrate that these "APO-skip " SSOs induce high levels of exon skipping and expression of the APOB87 isoform, but do not substantially inhibit APOB48 expression in cell lines. A single injection of an optimized APO-skip SSO into mice transgenic for human APOB resulted in abundant exon skipping that persists for >6 days. Weekly treatments generated a sustained reduction in LDL cholesterol levels of 34-51% in these mice, superior to pravastatin in a head-to-head comparison. These results validate APO-skip SSOs as a candidate therapy for FH.

Effects of dietary oxidized oil on laying performance, lipid metabolism, and apolipoprotein gene expression in laying hens

We studied the effects of dietary oxidized oils on serum lipid metabolic indices, estradiol level, and the gene expression of apolipoprotein B-100 and apolipoprotein VLDL-II in laying hens. Hy-Line Grey hens (280 ± 10 d old; average egg production, 90.0 ± 2.5%) were allotted to 1 of 4 dietary treatments, which were supplemented with 0 (control group), 1% (low oxidized group), 2% (moderately oxidized group), or 4% (highly oxidized group) thermally oxidized soybean oil. Each treatment contained 6 replicates, with 12 birds each. The feeding trial lasted for 30 d. Laying performance data were recorded weekly. Other indices were measured on d 0, 2, 6, 14, and 30 of the feeding trial. Hens in the moderately and highly oxidized groups had significantly lowered feed conversion ratios (P < 0.05). Those in the highly oxidized group also had decreased concentrations of serum very low density lipoprotein cholesterol on d 30 (P < 0.05) compared with the very low density lipoprotein cholesterol of hens in the moderately oxidized group. Hens in the moderately oxidized group had significantly increased expression of apolipoprotein B-100 (P < 0.05) from d 6 to 30. Consequently, hepatic triglyceride increased in this group on d 30 (P < 0.05). Serum triglyceride decreased in the moderately oxidized group on d 30 (P < 0.05), which may have been caused by the activation of peroxisome proliferator-activating receptor α. Serum estradiol levels were not significantly affected by oxidized oils at any time of measurement, but were significantly different between d 0 and 30 within the moderately oxidized group. This fact indicated that the effect of oxidized oils on apolipoprotein B-100 might partially be a cumulative result of the increasing secretion of estradiol. The results suggested that oxidized oil may affect the performance of laying hens through the regulation of apolipoproteins and estradiol.

Opposite Regulation of the Human Apolipoprotein M Gene by Hepatocyte Nuclear Factor 1 and Jun Transcription Factors

HDL is a negative risk factor for atherosclerosis because of its multiple atheroprotective functions. Inflammation converts HDL particles from anti-atherogenic to pro-atherogenic, and this transformation is associated with changes in HDL structure and composition. Apolipoprotein M (apoM) has been recently shown to play a role in the maturation of HDL in plasma and to protect from atherosclerosis. ApoM gene is expressed primarily in the liver and kidney and is down-regulated by pro-inflammatory signals. We now show that the human apoM promoter harbors a dual specificity regulatory element in the proximal region that binds hepatocyte nuclear factor 1 (HNF-1) and members of the AP-1 family of pro-inflammatory transcription factors (c-Jun and JunB). Overexpression of c-Jun or JunB repressed both the basal and the HNF-1-mediated transactivation of the human apoM promoter. Treatment of HepG2 cells with potent inflammation-inducing phorbol esters or overexpression of PKCα was associated with a marked inhibition of apoM gene expression in a c-Jun/JunB-dependent manner. We provide evidence for a novel mechanism of inflammation-induced transcriptional repression that is based on the competition between HNF-1 and Jun proteins for binding to the same regulatory region. A similar mechanism accounts for the down-regulation of the liver-specific apolipoprotein A-II gene by Jun factors. Our studies provide novel insights on the mechanisms that control the expression of liver-specific apolipoprotein genes during inflammation and could affect the maturation and the functionality of HDL particles.