Acylation-stimulating protein/C5L2-neutralizing antibodies alter triglyceride metabolism in vitro and in vivo

Acylation-stimulating protein (ASP), a lipogenic hormone, stimulates triglyceride (TG) synthesis and glucose transport upon activation of C5L2, a G protein-coupled receptor. ASP-deficient mice have reduced adipose tissue mass due to increased energy expenditure despite increased food intake. The objective of this study was to evaluate the blocking of ASP-C5L2 interaction via neutralizing antibodies (anti-ASP and anti-C5L2-L1 against C5L2 extracellular loop 1). In vitro, anti-ASP and anti-C5L2-L1 blocked ASP binding to C5L2 and efficiently inhibited ASP stimulation of TG synthesis and glucose transport. In vivo, neither anti-ASP nor anti-C5L2-L1 altered body weight, adipose tissue mass, food intake, or hormone levels (insulin, leptin, and adiponectin), but they did induce a significant delay in TG clearance [P < 0.0001, 2-way repeated-measures (RM) ANOVA] and NEFA clearance (P < 0.0001, 2-way RM ANOVA) after a fat load. After treatment with either anti-ASP or anti-C5L2-L1 antibody there was no change in adipose tissue AMPK activity, but neutralizing antibodies decreased perirenal TG mass (-38.4% anti-ASP, -18.8% anti-C5L2, P < 0.01-0.001) and perirenal LPL activity (-75.6% anti-ASP, -72.5% anti-C5L2, P < 0.05). In liver, anti-C5L2-L1 decreased TG mass (-42.8%, P < 0.05), whereas anti-ASP increased AMPK activity (+34.6%, P < 0.001). In the muscle, anti-C5L2-L1 significantly increased TG mass (+128.0%, P < 0.05), LPL activity (+226.1%, P < 0.001), and AMPK activity (+71.1%, P < 0.01). In addition, anti-ASP increased LPL activity (+164.4, P < 0.05) and AMPK activity (+53.9%, P < 0.05) in muscle. ASP/C5L2-neutralizing antibodies effectively block ASP-C5L2 interaction, altering lipid distribution and energy utilization.     

Inhibition of acylcoenzyme A:cholesterol acyltransferase activity in CaCo-2 cells results in intracellular triglyceride accumulation

The activity of acylcoenzyme A:cholesterol acyltransferase (ACAT) in CaCo-2 cells was inhibited by the ACAT inhibitor, 58-035. The inhibitory effect of this acylamide was specific for cholesterol esterification catalyzed by ACAT; the rates of triglyceride, phospholipid, and cholesterol synthesis were not inhibited by this agent. Cholesteryl esters were depleted in CaCo-2 cells 24 hr after inhibition of ACAT activity, whereas the unesterified cholesterol content increased by 56% after 96 hr. Moreover, inhibiting ACAT activity with 58-035 resulted in a time-dependent 2.5-fold increase in intracellular triglycerides. This accumulation of triglycerides in CaCo-2 cells was associated with a 37% increase in triglyceride synthesis by 96 hr in the presence of 58-035. Triglyceride-rich lipoprotein secretion (d less than 1.006 g/ml) was not affected by inhibiting ACAT activity for up to 6 hr. However, triglyceride-rich lipoprotein secretion was significantly decreased in CaCo-2 cells that were preincubated with 58-035 for 24 to 96 hr. Lipoproteins of density less than 1.006 g/ml that were isolated from CaCo-2 cells incubated with the ACAT inhibitor were deficient in cholesteryl esters and triglycerides compared to lipoproteins isolated from control cells. The data suggest that triglycerides accumulate in CaCo-2 cells in which ACAT activity has been inhibited by 58-035. This accumulation of triglycerides is associated with a modest increase in triglyceride synthesis and a decrease in triglyceride secretion. Altering intracellular cholesterol pools by regulating ACAT activity in the gut could result in the decrease of triglyceride transport and/or the secretion of triglyceride-rich lipoprotein particles of abnormal composition.     

Protoporphyrin IX accumulation disrupts mitochondrial dynamics and function in ABCG2-deficient hepatocytes

Targeted inhibition of multidrug ABCG2 transporter is believed to improve cancer therapeutics. However, the consequences of ABCG2 inhibition have not been systematically evaluated since ABCG2 is expressed in several organs including the liver. Here, we demonstrate that ABCG2-deficient hepatocytes have increased amounts of fragmental mitochondria accompanied by disruption of mitochondrial dynamics and functions. This disruption was due to ABCG2 knockout elevating intracellular protoporphyrin IX, which led to upregulation of DRP-1-mediated mitochondrial fission. The finding that ABCG2 deficiency can generate dysfunctional mitochondria in hepatocytes raises concerns regarding the systematic use of ABCG2 inhibitor in cancer patients.     

Dolichol: function, metabolism, and accumulation in human tissues.

Dolichol, a homologous series of alpha-saturated polyisoprenoid alcohols containing 14-24 isoprene units, was first isolated and characterized about 30 years ago. The phosphorylated form, dolichyl phosphate, is required for the biosynthesis of biologically important N-linked glycoproteins. Dolichol itself is synthesized by a common isoprenoid pathway from acetate and synthesis can be inhibited by some of the factors that inhibit cholesterol biosynthesis. It is metabolized very slowly and accumulates in tissues during aging and in certain lipid storage diseases. Dolichyl phosphate and cholesterol also accumulate in tissues during aging, but to a lesser extent than dolichol. Although dolichol and cholesterol have important metabolic functions, their accumulation in tissues can have deleterious effects.     

Effects of rice proteins from two cultivars, Koshihikari and Shunyo, on cholesterol and triglyceride metabolism in growing and adult rats

The effect and mechanism of two types of rice protein, one from regular japonica rice Koshihikari and another from rice cultivar Shunyo, with low glutelin and high prolamin content, on cholesterol and triglyceride metabolism were compared by feeding casein and soy protein to male Wistar strain rats 7 and 20 weeks old ad libitum for 2 weeks. The results in adult rats clearly indicated that both rice proteins had cholesterol-lowering effects in the plasma and the liver, comparable to soy protein, and the effects were accompanied with TG-lowering effects in the liver. Similar effects were also observed in growing rats when the diets were supplemented with cholesterol. The mechanism of the cholesterol-lowering effects by these rice proteins cannot be explained solely by fecal steroid excretion, but the results indicate that not only regular rice protein but also Shunyo rice protein possesses improving effects on lipid metabolism, especially in the adult period.     

Loss of sirtuin 1 and mitofusin 2 contributes to enhanced ischemia/reperfusion injury in aged livers

Ischemia/reperfusion (I/R) injury is a causative factor contributing to morbidity and mortality during liver resection and transplantation. Livers from elderly patients have a poorer recovery from these surgeries, indicating reduced reparative capacity with aging. Mechanisms underlying this age‐mediated hypersensitivity to I/R injury remain poorly understood. Here, we investigated how sirtuin 1 (SIRT1) and mitofusin 2 (MFN2) are affected by I/R in aged livers. Young (3 months) and old (23–26 months) male C57/BL6 mice were subjected to hepatic I/R in vivo. Primary hepatocytes isolated from each age group were also exposed to simulated in vitro I/R. Biochemical, genetic, and imaging analyses were performed to assess cell death, autophagy flux, mitophagy, and mitochondrial function. Compared to young mice, old livers showed accelerated liver injury following mild I/R. Reperfusion of old hepatocytes also showed necrosis, accompanied with defective autophagy, onset of the mitochondrial permeability transition, and mitochondrial dysfunction. Biochemical analysis indicated a near‐complete loss of both SIRT1 and MFN2 after I/R in old hepatocytes, which did not occur in young cells. Overexpression of either SIRT1 or MFN2 alone in old hepatocytes failed to mitigate I/R injury, while co‐overexpression of both proteins promoted autophagy and prevented mitochondrial dysfunction and cell death after reperfusion. Genetic approaches with deletion and point mutants revealed that SIRT1 deacetylated K655 and K662 residues in the C‐terminus of MFN2, leading to autophagy activation. The SIRT1‐MFN2 axis is pivotal during I/R recovery and may be a novel therapeutic target to reduce I/R injury in aged livers.     

Exogenous hypercholesterolemic rats, compared with their progenitor, Sprague-Dawley rats, promptly alter cholesterol metabolism in the liver and secrete cholesterol-rich particles in response to dietary cholesterol

Early responses of cholesterol metabolism to dietary cholesterol were compared between exogenous hypercholesterolemic (ExHC) and Sprague-Dawley rats. Both strains had a similar radioactivity of [¹⁴C]cholesterol in the serum half a day after the oral administration, but thereafter the radioactivity disappeared slowly in ExHC rats. ExHC rats promptly altered in response to the dietary cholesterol, activities of cholesterol 7α-hydroxylase and cholesterol synthesis in the liver and fecal excretion of bile acids derived from [¹⁴C]cholesterol administered orally. Lymphatic transport for 24 hr of [¹⁴C]cholesterol was similar between the strains. Triton administration resulted in a marked accumulation of cholesterol in serum d > 1.006 g/ml lipoproteins in ExHC rats; in addition, the formation of cholesteryl esters from [¹⁴C]oleic acid intravenously infused was greater in ExHC rats. These results indicate that ExHC rats increase serum cholesterol in response to exogenous cholesterol by decreasing the liver uptake and enhancing the secretion in the liver.     

The effect of natural and synthetic antioxidant polyhydroxynaphthoquinones on cholesterol metabolism in cultured rabbit hepatocytes]

Primary cultures of rabbit hepatocytes were used to examine the effect of natural and synthetic antioxidants--polyhydroxynaphthoquinones (PHNQ) and alpha-tocopherol on cholesterol and bile acid synthesis. Histochrome, one of the PHNQ, slightly decreased cholesterol synthesis at concentrations 10-100 microM, whereas alpha-tocopherol stimulated cholesterol synthesis. After administration of histochrome or alpha-tocopherol into culture medium a significant stimulation of bile acid synthesis in dose-dependent manner was observed. The increase of bile acid secretion by histochrome in the presence of physiological concentration of HDL2 was found as well. Since histochrome in contrast to alpha-tocopherol enhanced accumulation of [14C] cholesterol of HDL2 in the hepatocytes, it was concluded that histochrome stimulated bile acid synthesis as a result of increased input of HDL2 cholesterol into hepatocytes. These data suggest that histochrome may exhibit a hypocholesterolemic effect by stimulation of bile acid synthesis and inhibition of cholesterol synthesis.     

Overexpression of apolipoprotein AV in the liver reduces plasma triglyceride and cholesterol but not HDL in ApoE deficient mice

It has been shown that adenovirus-mediated overexpression of human ApoAV (hApoAV) in C57BL/6 mice results in decreased plasma triglyceride (TG) and total cholesterol (TC) levels with a major reduction occurring in the HDL fraction. In order to study the effect of ApoAV on hypercholesterolemic mice, an adenoviral vector expressing hApoAV was constructed and injected into ApoE deficient mice. High levels of hApoAV mRNA in the liver and ApoAV proteins in the liver and plasma were detected. The treatment reduced plasma TG levels by 50% and 75%, and TC levels by 45% and 58% at day 3 and 7, respectively, after treatment as compared with a control group treated with Ad-hAP (human alkaline phosphatase). Plasma HDL-C levels remained unaltered, which were different from normolipidemic mice. These findings suggest that ApoAV might serve as a therapeutic agent for hyperlipidemic disorder.     

Pharmacological activation of LXR in utero directly influences ABC transporter expression and function in mice but does not affect adult cholesterol metabolism

Cholesterol is critical for several cellular functions and essential for normal fetal development. Therefore, its metabolism is tightly controlled during all life stages. The liver X receptors-alpha (LXRalpha; NR1H3) and -beta (LXRbeta; NR1H2) are nuclear receptors that are of key relevance in coordinating cholesterol and fatty acid metabolism. The aim of this study was to elucidate whether fetal cholesterol metabolism can be influenced in utero via pharmacological activation of LXR and whether this would have long-term effects on cholesterol homeostasis. Administration of the LXR agonist T0901317 to pregnant mice via their diet (0.015% wt/wt) led to induced fetal hepatic expression levels of the cholesterol transporter genes Abcg5/g8 and Abca1, higher plasma cholesterol levels, and lower hepatic cholesterol levels compared with controls. These profound changes during fetal development did not affect cholesterol metabolism in adulthood nor did they influence coping with a high-fat/high-cholesterol diet. This study shows that the LXR system is functional in fetal mice and susceptible to pharmacological activation. Despite massive changes in fetal cholesterol metabolism, regulatory mechanisms involved in cholesterol metabolism return to a "normal" state in offspring and allow coping with a high-fat/high-cholesterol diet.     

Effect of mevinolin and glycyrrhizinic acid on cholesterol and bile acid metabolism in cultured rabbit hepatocytes]

A comparison of effects of two hypocholesterolemic drugs--mevinolin and glycyrrhizinic acid, on cholesterol and bile acid metabolism in cultured rabbit hepatocytes has been carried out. The following parameters have been determined: i) cholesterol synthesis from [2-14C]acetate; ii) bile acid production from newly synthesized and [4-14C]-labeled HDL2 cholesterol, and, iii) total cholesterol efflux into the incubation medium Mevinolin (0.5 microgram/ml) inhibited [2-14C] acetate incorporation into cholesterol by more than 90%. Conversely, glycyrrhizinic acid did not influence cholesterol synthesis even when used at high (100 micrograms/ml) concentrations but stimulated the conversion of endogenous (by 37%) and exogenous (by 18%) cholesterol into bile acids and increased, in addition, the proportion of bile acids in the total sterol pool released from hepatocytes into the incubation medium. At the same time, mevinolin used at 0.5 microgram/ml decreased the bile acid production by endogenous (by 27%) and exogenous (by 40%) cholesterol. The data obtained suggest that glycyrrhizinic acid exerts hypocholesterolemic action by stimulation of cholesterol conversion into bile acids without any effect on cholesterol synthesis. As for mevinolin, it has a cholesterol-suppressing effect via a mechanism of cholesterol synthesis inhibition only.     

Health-promoting effects of bovine colostrum in Type 2 diabetic patients can reduce blood glucose, cholesterol, triglyceride and ketones

Bovine colostrum (BC) has been reported to enhance immune function, reduce fat accumulation and facilitate the movement of glucose to the muscle. However, very few attempts have been made to examine its anti-diabetic effects in diabetes patients. The aim of this study was to evaluate whether BC decreases blood glucose, as well as cholesterol, triglyceride (TG) and ketones levels, which can be elevated by obesity and stress in Type 2 diabetic patients. Sixteen patients (men=8, women=8) with Type 2 diabetes were randomized into the study. Each ingested 5 g of BC on an empty stomach every morning and night for 4 weeks. Blood glucose, ketones (beta-hydroxybutyric acid), total cholesterol and TGs were measured every week. In both the men and women, blood glucose levels at 2 and 8 h postprandial decreased continually during the experimental period. The rate of decrease in blood glucose at 8 h postprandial was not different between the men and women, but was higher in the women (14.25+/-2.66) than in the men (10.96+/-1.82%) at 2 h postprandial. Total cholesterol and TG levels decreased significantly in both the men and women after 4 weeks. Also, beta-hydroxybutyric acid level decreased with BC ingestion, but this was not significant. These results suggest that BC can decrease levels of blood glucose and ketones, as well as reduce cholesterol and TGs, all of which may cause complications in Type 2 diabetic patients.     

Vitamin E and caloric restriction promote hepatic homeostasis through expression of connexin 26, N-cad,E-cad and cholesterol metabolism genes

Connexins (Cx) and cadherins are responsible for cell homeostasis. The Cx activity is directly related to cholesterol. The present work investigates whether vitamin E, with or without caloric restriction (CR), alters the mRNA expression of Cx26, Cx32, Cx43, N-cadherins (N-cads), E-cadherins (E-cads) and alpha-smooth muscle actin (α-SMA), and evaluates their relation to cholesterol metabolism in rat liver. Animals were divided into different groups: control with ad libitum diet (C), control+vitamin E (CV), aloric restriction with intake to 60% of group C (CR), and the intake of group CR+vitamin E (RV). There were increases of manganese superoxide dismutase (Mn-SOD) and glutathione S-transferase mu 1, indicating antioxidant effects of CR and vitamin E. An increase of nitric oxide in the CR group was in agreement with the Mn-SOD data. Supplementation with vitamin E, with or without CR, upregulated the expression of Cx26 mRNA and increased low-density lipoprotein cholesterol (LDL-c) in the CV group. Reductions of Cx32 and Cx43 were associated with lower LDL-c. Increases in Hmgcr and low-density lipoprotein receptor (LDLr) in the CV and RV groups could be explained by the effect of vitamin E. A reduction of LDLr in the CR group was due to the reduced dietary intake. Increases in cadherins in the CV, CR and RV groups were indicative of tissue maintenance, which was also supported by increases of α-SMA in groups CV and RV. Finally, vitamin E, with or without CR, increased Cx26, probably modulated by expression of the Hmgcr and LDLr genes. This suggests important relationship of Cxs and cholesterol metabolism genes.     

Control of cholesterol biosynthesis,uptake and storage in hepatocytes by Cideb

Cideb, a member of CIDE family proteins, has emerged as an important regulator in the development of obesity and diabetes by controlling fatty acid synthesis and VLDL secretion in hepatocytes. Here, we investigated the role of Cideb in cholesterol biosynthesis, uptake and storage in the liver by using Cideb-null mice as a model system. Cideb-null mice and wild-type mice were treated with normal diet (ND) or high cholesterol diet (HCD) for one month. The metabolic parameters of cholesterol metabolism and expression profiles of genes in cholesterol biosynthesis and storage were measured. Cideb-null mice had lower levels of plasma cholesterol and LDL when fed with both ND and HCD and increased rate of cholesterol absorption. Furthermore, the liver of Cideb-null mice has lower rates of cholesterol biosynthesis and reduced expression levels of sterol response element-binding protein (SREBP) cleavage-activation protein (SCAP), and lower levels of nuclear form of SREBP2 and its downstream target genes in cholesterol biosynthesis pathway under a normal diet treatment. On the contrary, hepatic cholesterol biosynthesis rate between wild-type and Cideb-null mice was similar after high cholesterol diet treatment. Interestingly, hepatic cholesterol storage in the liver of Cideb-null mice was significantly increased due to its increased LDL receptor (LDLR) and acyl-CoA cholesterol acyltransferase (ACAT) expression. Finally, we observed drastically reduced cholesterol levels in the heart of Cideb-null mice fed with a high cholesterol diet. Overall, our data suggest that Cideb is a novel regulator in controlling cholesterol homeostasis in the liver. Therefore, Cideb could serve as an important therapeutical target for the treatment of atherosclerosis and cardiovascular diseases.     

Chromosomal localization of genes involved in biosynthesis,metabolism or transport of cholesterol in the rat

Several genes involved in biosynthesis, transport or metabolism of cholesterol have been localized on rat chromosomes by using a radiation hybrid (RH) panel. The genes, coding for squalene epoxidase (Sqle), mevalonate kinase (Mvk), and farnesyl diphosphate farnesyl transferase 1 (Fdft1) which are involved in cholesterol biosynthesis, have been mapped on chromosome 7, 12, and 15, respectively. The genes coding for phospholipid transfer protein (Pltp), sterol carrier protein-2 (Scp2), ATP binding cassette reporter A7 (Abca7), scavenger receptor class B, type 1 (Cd36l1), steroidogenic acute regulatory protein (Star), and lecithin:cholesterol acyl transferase (Lcat), which are involved in the transfer and/or metabolism of cholesterol, have been mapped on chromosome 3, 5, 7, 12, 16, and 19, respectively. Each of the genes Scp2, Sqle and Fdft1 maps close to a QTL for serum total cholesterol in rat, suggesting that these three genes might represent candidate genes for the previously mapped QTLs.