LDLR Levels Research Articles

Relationships between di-(2-ethylhexyl) phthalate exposure and lipid metabolism in adolescents: Human data and experimental rat model analyses

In recent years, the incidence of lipid metabolism disorders in adolescents has gradually increased, and the effects of DEHP on lipid metabolism have received widespread attention. In this study, 463 adolescents aged 16–19 years were enrolled as subjects. This study analyzed the associations between the urinary levels of DEHP metabolites (MEHP, MEOHP, MEHHP, MECPP, MCMHP, and ∑DEHP) and BMI, WHR, WtHR, VAI, LAP, the plasma levels of lipids (TC, TG, HDL-C, and LDL-C), and the peripheral blood leukocyte mRNA levels of SREBP-2, SR-BI, LDLR, and NR1H3. Animal experiments were performed to confirm and expand findings. Wistar rats were administered DEHP at 0, 5, 50, and 500 mg/kg/d for 8 weeks. The serum and liver levels of TC, TG, HDL-C, and LDL-C, and the liver mRNA and protein levels of SREBP-2, SR-BI, LDLR, and NR1H3 were measured. The results showed that WHR, VAI, and LAP were significantly positively associated with the urinary levels of MECPP and ∑DEHP; the plasma HDL-C level was significantly negatively associated with the levels of MECPP, MCMHP and ∑DEHP; the peripheral blood leukocyte mRNA levels of SREBP-2, NR1H3, and LDLR were significantly positively correlated with the MCMHP level; and the SR-BI mRNA level was significantly positively correlated with the levels of MECPP and MCMHP in adolescents. Moreover, the results of animal experiments showed that DEHP exposure significantly increased the serum levels of TC, HDL-C, and LDL-C in 500 mg/kg/d group, as well as the liver levels of TC and HDL-C, up-regulated SREBP-2 mRNA and protein expression in 50 and 500 mg/kg/d groups. DEHP exposure significantly down-regulated SR-BI and NR1H3 protein expression in the liver of the 500 mg/kg/d group rats. Our findings indicate that DEHP exposure can affect lipid metabolism in adolescents by regulating the expression of lipid metabolism-related genes.

Patterns and tempo of PCSK9 pseudogenizations suggest an ancient divergence in mammalian cholesterol homeostasis mechanisms

Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a central role in cholesterol homeostasis in humans as a major regulator of LDLR levels. PCSK9 is an intriguing protease in that it does not act by proteolysis but by preventing LDLR recirculation from endosomes to the plasma membrane. This, and the inexistence of any other proteolytic substrate but itself could suggest that PCSK9 is an exquisite example of evolutionary fine-tuning. However, the gene has been lost in several mammalian species, and null alleles are present (albeit at low frequencies) in some human populations without apparently deleterious health effects, raising the possibility that the PCSK9 may have become dispensable in the mammalian lineage. To address this issue, we systematically recovered, assembled, corrected, annotated and analysed publicly available PCSK9 sequences for 420 eutherian species to determine the distribution, frequencies, mechanisms and timing of PCSK9 pseudogenization events, as well as the evolutionary pressures underlying the preservation or loss of the gene. We found a dramatic difference in the patterns of PCSK9 retention and loss between Euarchontoglires—where there is strong pressure for gene preservation—and Laurasiatheria, where multiple independent events have led to PCSK9 loss in most species. These results suggest that there is a fundamental difference in the regulation of cholesterol metabolism between Euarchontoglires and Laurasiatheria, which in turn has important implications for the use of Laurasiatheria species (e.g. pigs) as animal models of human cholesterol-related diseases.

Construction and application of a human scFv phage display library based on Cre‑LoxP recombination for anti‑PCSK9 antibody selection.

A large human natural single-chain fragment variable (scFv) phage library was constructed based on Cre-LoxP recombination, and used to successfully identify antibodies against proprotein convertase subtilisin/kexin type 9 (PCSK9). The library was derived from 400 blood samples, 30 bone marrow samples, and 10 cord blood samples from healthy donors. Lymphocytes were isolated from each sample and cDNA was synthesized using reverse transcription-quantitative PCR. Two-step overlap PCR was then used for scFv synthesis using a LoxP peptide as the linker. The scFv gene was inserted into the phagemid vector pDF by enzymatic digestion and ligation, and then transformed into Escherichia coli (E. coli) SS320 to establish a primary antibody library in the form of scFvs. A primary antibody library consisting of 5×107 peripheral blood and umbilical cord blood sources, as well as a primary antibody library of 5×107 bone marrow samples were obtained. By optimizing the recombination conditions, the primary phage library was used to infect E. coliBS1365 strain (which expresses the Cre enzyme), and a human scFv recombinant library with a size of 1×1011 was obtained through Cre-LoxP enzyme-mediated heavy and light chain replacement and recombination. This constructed recombinant library was employed to screen for antibodies against recombinant PCSK9. After four rounds of selection, a fully human antibody (3D2) was identified with a binding affinity of 1.96±1.56ⅹ10−10 M towards PCSK9. In vitro, the PCSK9/low-density lipoprotein receptor (LDLR) pathway of Hep-G2 cells was inhibited by 3D2 treatment, thereby increasing LDL uptake in these cells. In addition, combination treatment with 3D2 and statin was more effective at increasing LDLR levels than treatment with 3D2 or statin alone. Furthermore, 3D2 resulted in a 3-fold increase in hepatic LDLR levels, and lowered total serum cholesterol by up to 61.5% in vivo. Taken together, these results suggest that the constructed human Cre-LoxP scFv phage display library can be used to screen fully human scFv, and that 3D2 may serve as a candidate hypolipidemic therapy.

High Cholesterol Diet Exacerbates Blood-Brain Barrier Disruption in LDLr-/- Mice: Impact on Cognitive Function.

Background:Evidence has revealed an association between familial hypercholesterolemia and cognitive impairment. In this regard, a connection between cognitive deficits and hippocampal blood-brain barrier (BBB) breakdown was found in low-density lipoprotein receptor knockout mice (LDLr–/–), a mouse model of familial hypercholesterolemia.Objective:Herein we investigated the impact of a hypercholesterolemic diet on cognition and BBB function in C57BL/6 wild-type and LDLr–/–mice.Methods:Animals were fed with normal or high cholesterol diets for 30 days. Thus, wild-type and LDLr–/–mice were submitted to memory paradigms. Additionally, BBB integrity was evaluated in the mice’s prefrontal cortices and hippocampi.Results:A tenfold elevation in plasma cholesterol levels of LDLr–/–mice was observed after a hypercholesterolemic diet, while in wild-type mice, the hypercholesterolemic diet exposure increased plasma cholesterol levels only moderately and did not induce cognitive impairment. LDLr–/–mice presented memory impairment regardless of the diet. We observed BBB disruption as an increased permeability to sodium fluorescein in the prefrontal cortices and hippocampi and a decrease on hippocampal claudin-5 and occludin mRNA levels in both wild-type and LDLr–/–mice treated with a hypercholesterolemic diet. The LDLr–/–mice fed with a regular diet already presented BBB dysfunction. The BBB-increased leakage in the hippocampi of LDLr–/–mice was related to high microvessel content and intense astrogliosis, which did not occur in the control mice.Conclusion:Therefore, LDLr–/–mice seem to be more susceptible to cognitive impairments and BBB damage induced by exposure to a high cholesterol diet. Finally, BBB disruption appears to be a relevant event in hypercholesterolemia-induced brain alterations.

Srebf2 Locus Overexpression Reduces Body Weight, Total Cholesterol and Glucose Levels in Mice Fed with Two Different Diets.

Macronutrients represent risk factors for hyperlipidemia or diabetes. Lipid alterations and type 2 diabetes mellitus are global health problems. Overexpression of sterol regulatory element-binding factor (Srebf2) in transgenic animals is linked to elevated cholesterol levels and diabetes development. We investigated the impact of increased Srebf2 locus expression and the effects of control and high-fat, high-sucrose (HFHS) diets on body weight, glucose and lipid metabolisms in transgenic mice (S-mice). Wild type (WT) and S-mice were fed with both diets for 16 weeks. Plasma glucose, insulin and lipids were assessed (n = 25). Immunostainings were performed in liver, pancreas and fat (N = 10). Expression of Ldlr and Hmgcr in liver was performed by RT-PCR (N = 8). Control diet: S-mice showed reduced weight, insulin, total and HDL cholesterol and triglycerides (TG). HFHS diet widened differences in weight, total and HDL cholesterol, insulin and HOMA index but increased TG in S-mice. In S-mice, adipocyte size was lower while HFHS diet produced lower increase, pancreatic β-cell mass was lower with both diets and Srebf2, Ldlr and Hmgcr mRNA levels were higher while HFHS diet produced a rise in Srebf2 and Hmgcr levels. Srebf2 complete gene overexpression seems to have beneficial effects on metabolic parameters and to protect against HFHS diet effects.

Fermented Rhus Verniciflua Stokes Extract Alleviates Nonalcoholic Fatty Liver through the AMPK/SREBP1/PCSK9 Pathway in HFD-Induced Nonalcoholic Fatty Liver Animal Model

Background: We have previously reported the anti-hepatic lipogenic effect of fermented Rhus verniciflua stokes extract (FRVE) in an oleic-acid-treated HepG2 cell model. Methods: Herein, we advanced our understanding and evaluated the impact of FRVE in HFD-fed C57BL/6 mice using an animal model of nonalcoholic fatty liver disease (NAFLD). Milk thistle extract was used as a positive control to compare the effects of FRVE. Results: FRVE decreased body weight, intra-abdominal fat weight, and liver weight. Furthermore, FRVE decreased HFD-induced elevated serum levels of ALT, AST, TC, and TG, and downregulated the increase in hepatic lipid accumulation and TG levels. FRVE reduced hepatic SREBP-1, PCSK-9, SREBP-2, and ApoB mRNA levels. IHC data showed that FRVE reduced the levels of nucleic SREBP-1, increased the levels of LDLR, and upregulated the expression of p-AMPK. Conclusion: Overall, these results demonstrate the anti-hepatic lipidemic effect of FRVE in an animal model. These findings are consistent with our previous study and strongly suggest that FRVE exerts anti-hepatic lipogenic effects by activating AMPK.

Triphenyltin chloride reduces the development of rat adrenal cortex during puberty

Triphenyltin has been classified as an endocrine disruptor. However, whether triphenyltin interferes with the adrenal glands during puberty remains unknown. Here, we reported the effects of triphenyltin on the adrenal glands in rats. Male Sprague Dawley rats (age of 35 days) were orally administered with 0, 0.5, 1, or 2 mg/kg/day triphenyltin for 18 days. Triphenyltin significantly lowered corticosterone levels at 1 and 2 mg/kg and adrenocorticotropic hormone at 2 mg/kg. The RNA-Seq analysis detected multiple differentially expressed genes. Four down-regulated genes were transcription factor genes (Nr4a1, Nr4a2, Nr4a3, and Ppard), which might be associated with the suppression of the adrenal cortex function. RNA-seq and qPCR showed that triphenyltin dose-dependently down-regulated the expression of the genes for cholesterol transport and biosynthesis, including Scarb1, Ldlr, Hmgcs1, Hmgcr, and Hsd17b7. Further Western blotting revealed that it lowered NR4A1, PPRAD, LDLR, and HMGCS1 protein levels. We treated H295R adrenal cells with 1–100 nM triphenyltin for 72 h. Triphenyltin induced significant higher ROS production at 100 nM and did not induce apoptosis at 10 and 100 nM. In conclusion, triphenyltin inhibits production of corticosterone via blocking the expression of cholesterol uptake transporters and cholesterol biosynthesis.

Biotransformation of ginsenoside Rb1 with wild Cordyceps sinensis and Ascomycota sp. and its antihyperlipidemic effects on the diet-induced cholesterol of zebrafish.

Biotransformation major ginsenoside into minor ginsenoside via microbial fermentation has been proposed as a viable option to produce minor ginsenoside, becauseof its biological activity superior to major ginsenoside. Cordyceps sinensis contains a complex enzymatic system and many ingredients with medicinal value that could be useful tools for biotransformation applications in the ginseng industry. Wild C. sinensis and Ascomycota sp. were collected from Changbai Mountain and identified. Analysis by UPLC-MS and HPLC indicates that the underlying pathway of major ginsenoside Rb1 during fermentation with strains was Rb1→Rd→F2→CK and Rb1→Rd→Rg3. C. sinensis and Ascomycota sp. can be applied to minor ginsenoside preparation in the food and medical industries. The antihyperlipidemic effects of Rb1 were further screened from fermentation in larvae zebrafish based on the fluorescence intensity. In the adult zebrafish model, treatment with high-dose ginsenoside Rb1 group exhibited a significant decrease in the plasma total cholesterol (TC) and triglyceride (TG) levels by 36.49% (p<.05) and 29.97% (p<.05), respectively, compared with high cholesterol group (HC). Furthermore, ginsenoside Rb1 treatment decreased the mRNA levels of LDLR and SREBP2 in the adult zebrafish liver. Ginsenoside Rb1 diet supplement significantly increased the mRNA expression of HMGCR and CYP7A1. These results suggest that ginsenoside Rb1 attenuates hypercholesterolemia via the downregulation of cholesterol synthesis and assembly or secretion of lipoproteins as well as the upregulation of cholesterol transport and efflux, providing a novel idea of ginsenoside keeping cholesterol levels down for the clinical application. PRACTICAL APPLICATIONS: Wild Cordyceps sinensis has the potential to be applied to the preparation for minor ginsenoside. Furthermore, the final fermentation product has more functional characteristics, including cordyceps acid, cordycepin, and adenosine. Wild Cordyceps sinensis and Ascomycota sp. could potentially be employed in the food and medical industries.

17β-Estradiol Inhibits PCSK9-Mediated LDLR Degradation Through GPER/PLC Activation in HepG2 Cells.

Plasma levels of PCSK9 are significantly higher in postmenopausal women. Pharmacologically increased estrogen levels have been shown to lower PCSK9 and LDL-C levels in animals and humans. The action of estrogen suggests that it has the ability to prevent PCSK9-mediated LDLR degradation in liver cells. However, little is known about how estrogen alters PCSK9-mediated LDLR degradation. Here, we report that 17β-estradiol (βE2) reduces PCSK9-mediated LDLR degradation by a mechanism that involves activation of the G protein-coupled estrogen receptor (GPER). In cultured HepG2 cells, βE2 prevented the internalization of PCSK9, which subsequently lead to PCSK9-mediated LDLR degradation. The altered LDLR levels also resulted in an increase in LDL uptake that was not observed in the absence of PCSK9. In addition, we showed that clathrin was rapidly increased in the presence of PCSK9, and this increase was blocked by βE2 incubation, suggesting rapid recruitment of clathrin in HepG2 cells. PLCγ activation and intracellular Ca2+ release were both increased due to the rapid effect of estrogen. By using a GPER antagonist G15, we demonstrated that the GPER mediates the action of estrogen. Together, the data from this in vitro study demonstrate that estrogen can regulate LDLR levels mainly through GPER activation, which prevents PCSK9-dependent LDLR degradation in HepG2 cells.

Gestational hypothyroidism elicits more pronounced lipid dysregulation in mice than pre-pregnant hypothyroidism.

Thyroid hormone is crucial for regulating lipid and glucose metabolism, which plays essential role in maintaining the health of pregnant women and their offspring. However, the current literature is just focusing on the development of offspring born to the untreated mothers with hypothyroidism, rather than mothers themselves. Additionally, the interaction between hypothyroidism and pregnancy, and its impact on the women's health are still elusive. Therefore, this study was designed to compare the metabolic differences in dams with hypothyroidism starting before pregnancy and after pregnancy. Pre-pregnant hypothyroidism was generated in 5-week-old female C57/BL/6J mice using iodine-deficient diet containing 0.15% propylthiouracil for 4 weeks, and the hypothyroidism was maintained until delivery. Gestational hypothyroidism was induced in dams after mating, using the same diet intervention until delivery. Compared with normal control, gestational hypothyroidism exhibited more prominent increase than pre-pregnant hypothyroidism in plasma total cholesterol and low-density lipoprotein cholesterol, and caused hepatic triglycerides accumulation. Similarly, more significant elevations of protein expressions of SREBP1c and p-ACL, while more dramatic inhibition of CPT1A and LDL-R levels were also observed in murine livers with gestational hypothyroidism than those with pre-pregnant hypothyroidism. Moreover, the murine hepatic levels of total cholesterol and gluconeogenesis were dramatically and equally enhanced in two hypothyroid groups, while plasma triglycerides and protein expressions of p-AKT, p-FoxO1 and APOC3 were reduced substantially in two hypothyroid groups. Taken together, our current study illuminated that gestational hypothyroidism may elicit more pronounced lipid dysregulation in dams than dose the pre-pregnant hypothyroidism.

Interleukin 10 promotes macrophage uptake of HDL and LDL by stimulating fluid-phase endocytosis

ObjectiveHighly elevated plasma levels of interleukin-10 (IL-10) are causally associated with “Disappearing HDL Syndrome” and low plasma LDL-cholesterol, but the underlying mechanism is poorly understood. Fluid-phase endocytosis, a process highly dependent on actin dynamics, enables cells to internalize relatively high amounts of extracellular fluids and solutes. We sought to investigate whether IL-10 induces lipoprotein uptake by fluid-phase endocytosis in macrophages. Methods and resultsMacrophages (RAW264.7, Kupffer and human) were incubated with vehicle (PBS) or IL-10 (20 ng/ml) for 7 days. Uptake of HDL, LDL, and/or fluid-phase endocytosis probes (albumin-Alexa680®, 70 kDa FITC-Dextran and Lucifer Yellow, LY) was evaluated by FACS. Intracellular cofilin and phosphorylated cofilin (p-cofilin) levels were determined by immunoblotting.Macrophage uptake of lipoproteins and probes was non-saturable and increased after IL-10 incubation (p < 0.0001). Furthermore, pre-incubation with fluid-phase endocytosis inhibitors (LY294002, Latrunculin A, and Amiloride) significantly reduced uptake (p < 0.05). IL-10 increased the cofilin/p-cofilin ratio (p = 0.021), signifying increased cofilin activation and hence filamentous actin. Consistently, phalloidin staining revealed increased filamentous actin in macrophages after IL-10 treatment (p = 0.0018). Finally, RNA-seq analysis demonstrated enrichment of gene sets related to actin filament dynamics, membrane ruffle formation and endocytosis in IL-10-treated macrophages (p < 0.05). IL-10 did not alter mRNA levels of Ldlr, Vldlr, Scarb1, Cd36 or Lrp1. In primary human monocyte-derived macrophages and murine Kupffer cells, IL-10 incubation also increased uptake of lipoproteins, albumin and LY (p < 0.01). ConclusionsInterleukin-10 induces the uptake of HDL and LDL by fluid-phase endocytosis by increasing actin-filament rearrangement in macrophages, thus providing a plausible mechanism contributing to “Disappearing HDL Syndrome”.

High-Fructose Diet-Induced Hypertriglyceridemia Is Associated With Enhanced Hepatic Expression of ACAT2 in Rats.

High levels of fructose induce hypertriglyceridemia, characterized by excessive levels of triglyceride-rich lipoproteins such as very low-density lipoprotein (VLDL); however, the underlying mechanisms are poorly understood. The aim of this short communication was to examine hepatic changes in the expression of genes related to cholesterol metabolism in rats with hypertriglyceridemia induced by high-fructose or high-glucose diets. Rats were fed a 65 % (w/w) glucose diet or a 65 % (w/w) fructose diet for 12 days. Serum levels of triglycerides, total cholesterol, and VLDL+LDL-cholesterol, hepatic levels of triglycerides and cholesterol, and ACAT2 expression at the gene and protein levels were significantly higher in the fructose diet group compared to the glucose diet group. The hepatic levels of Abcg5/8 were lower in the fructose group than in the glucose group. Serum high-density lipoprotein (HDL)-cholesterol and hepatic expression levels of Hmgcr, Ldlr, Acat1, Mttp, Apob, and Cyp7a1 did not differ significantly between groups. These findings suggest that high-fructose diet-induced hypertriglyceridemia is associated with increased hepatic ACAT2 expression.

A common variant in CCDC93 protects against myocardial infarction and cardiovascular mortality by regulating endosomal trafficking of low-density lipoprotein receptor.

Genome-wide association studies have previously identified INSIG2 as a candidate gene for plasma low-density lipoprotein cholesterol (LDL-c). However, we suspect a role for CCDC93 in the same locus because of its involvement in the recycling of the LDL-receptor (LDLR). Characterization of the INSIG2 locus was followed by studies in over 107000 individuals from the general population, the Copenhagen General Population Study and the Copenhagen City Heart Study, for associations of genetic variants with plasma lipids levels, with risk of myocardial infarction (MI) and with cardiovascular mortality. CCDC93 was furthermore studied in cells and mice. The lead variant of the INSIG2 locus (rs10490626) is not associated with changes in the expression of nearby genes but is a part of a genetic block, which excludes INSIG2. This block includes a coding variant in CCDC93 p.Pro228Leu, which is in strong linkage disequilibrium with rs10490626 (r2 > 0.96). In the general population, separately and combined, CCDC93 p.Pro228Leu is dose-dependently associated with lower LDL-c (P-trend 2.5 × 10-6 to 8.0 × 10-9), with lower risk of MI (P-trend 0.04-0.002) and lower risk of cardiovascular mortality (P-trend 0.005-0.004). These results were validated for LDL-c, risk of both coronary artery disease and MI in meta-analyses including from 194000 to >700000 participants. The variant is shown to increase CCDC93 protein stability, while overexpression of human CCDC93 decreases plasma LDL-c in mice. Conversely, CCDC93 ablation reduces LDL uptake as a result of reduced LDLR levels at the cell membrane. This study provides evidence that a common variant in CCDC93, encoding a protein involved in recycling of the LDLR, is associated with lower LDL-c levels, lower risk of MI and cardiovascular mortality.

Regulation of triglyceride synthesis by estradiol in the livers of hybrid tilapia (Oreochromis niloticus ♀ × O. aureus ♂).

Estradiol (E2) is a sex steroid hormone that modulates multiple physiological processes in teleosts. The aim of this study was to explore the role of E2 in the hepatic lipid metabolism of hybrid tilapia. The hybrid tilapias were injected with different concentrations of E2 (0 mg/kg, 10 mg/kg, 25 mg/kg and 50 mg/kg) and ICI 182,780 (ICI) (35 mg/kg) (an E2 receptor antagonist). Subsequently, the liver lipid depositions were analyzed by tissue sections with oil red O staining. Serum total cholesterol (TC), triglyceride (TG), high-density lipoprotein (HDL), low-density lipoprotein (LDL) and nonesterified fatty acids (NEFAs) were assayed from the fish in different groups. Genes related to very low-density lipoprotein (VLDL) assembly, lipoprotein lipase and lipoprotein receptors, fatty acid uptake and triacylglycerol metabolism were determined by quantitative RT-PCR. The results showed that 50 mg/kg E2 injections enlarged the lipid droplets significantly. Simultaneously, the E2 injections tended to upregulate TC, TG, LDL, and HDL in the serum. The 50 mg/kg E2 group showed a significantly higher expression of the VLDL assembly genes but depressed levels of LDLR and LRP1. In addition, FABP3, FABP11a and DGAT2 were significantly elevated, while CD36 and ACO1 decreased in the 50 mg/kg E2 injection. The ICI injection inhibited the expression of MTP, LPL, LRP1, CD36, FABP11a, ACO1 and FAS in tilapia livers. These results demonstrated that by stimulating the expression of genes associated with the VLDL assembly, inhibiting lipoprotein lipase and lipoprotein receptor-related genes and promoting the rate-limiting enzyme in the synthesis of the TG, E2 induced deposition of lipids in the livers of hybrid tilapia. Overall, the results suggest a role for E2 in fish lipid metabolisms that provide new clues to illustrate the sex steroid function in energy metabolism in livers.

Dietary apple polyphenols supplementation enhances antioxidant capacity and improves lipid metabolism in weaned piglets.

Apple polyphenols (APPs) are biologically active flavonoids that have antioxidant, anti-inflammatory, improving insulin sensitivity, hypocholesterolaemic effect and antiviral properties. This study was conducted to explore effects of dietary APPs supplementation on antioxidant activities and lipid metabolism in weaned piglets. Fifty-four weaned piglets (half male and female) were randomly divided into three groups with six replicates in each group and three piglets in each repetition. Piglets were fed control diet (basal diet) or a control diet supplemented with 400mg/kg or 800mg/kg APPs for 6weeks. Blood and liver samples were collected to determine biochemical, antioxidant and lipid metabolism parameters. Here we showed that dietary APPs supplementation increased HDL-C and decreased T-CHO, TG and LDL-C concentrations. Dietary APPs supplementation increased antioxidative capacity in serum and CAT activity in liver, and significantly increased the mRNA expressions of CAT, GST and SOD1 in liver. ACC mRNA level and LPL activity were tended to decrease by APPs. HMG-CoAR, CTP7A1, CD36 and FATP1 mRNA levels were decreased by APPs, while LDL-R, PGC-1α, Sirt1 and CPT1b mRNA levels were increased by 400mg/kg APPs. No alterations in growth performance were found in all treatments. This study firstly provided the evidence that dietary APPs supplementation could enhance systemic antioxidant capacity and improve lipid metabolism in weaned piglets. The mechanism by which APPs improve lipid metabolism might be through regulating hepatic cholesterol metabolism and increasing fatty acid oxidation, and decreasing fatty acid uptake and de novo synthesis.

Clinical relevance of cholesterol homeostasis genes in colorectal cancer

Accumulation of cholesterol is a well-known feature in cancer. Preclinical studies suggest the contribution of various cholesterol regulators in CRC. However, their clinical relevance remains poorly understood. The aim of the present study is to evaluate the expression of these modulators in CRC and elucidate their diagnostic and prognostic value. mRNA levels of HMGCR, SREBF2, NR1H3 and NR1H2 were downregulated in tumors in local and TCGA cohort. The expression of LDLR, ABCA1 and SCARB1 was not consistent in the two cohorts. Western Blot analysis showed the increased levels of LDLR and reduced levels of LXR in early stage patients. Tumoral SREBP2 levels were enhanced in early stage whereas decreased in late stage. The individual expression of HMGCR, SREBF2, NR1H3 and NR1H2 did not have the potential to be used as independent prognostic marker, however, the combined expression of these genes associated with poor clinical outcome independent of lymph node metastasis, distant metastasis and advanced stage. This work sheds light on deregulation of cholesterol uptake and efflux pathways and provides novel leads in the development of biomarkers and therapeutic regimens that can detect and target CRC at initial stages.

Using metabolic profiling and gene expression analyses to explore molecular effects of replacing saturated fat with polyunsaturated fat—a randomized controlled dietary intervention study

ABSTRACTBackgroundReplacing dietary saturated fatty acids (SFAs) with polyunsaturated fatty acids (PUFA) reduces the plasma low-density lipoprotein (LDL) cholesterol and subsequently the risk of cardiovascular disease. However, beyond changes in LDL cholesterol, we lack a complete understanding of the physiologic alterations that occur when improving dietary fat quality.ObjectivesThe aim of this study was to gain knowledge of metabolic alterations paralleling improvements in the fat quality of the diet.MethodsWe recently conducted an 8-wk, double-blind, randomized controlled trial replacing SFAs with PUFAs in healthy subjects with moderate hypercholesterolemia (n = 99). In the present substudy, we performed comprehensive metabolic profiling with multiple platforms (both nuclear magnetic resonance- and mass spectrometry-based technology) (n = 99), and analyzed peripheral blood mononuclear cell gene expression (n = 95) by quantitative real-time polymerase chain reaction.ResultsA large number of lipoprotein subclasses, myristoylcarnitine and palmitoylcarnitine, and kynurenine were reduced when SFAs were replaced with PUFAs. In contrast, bile acids, proprotein convertase subtilisin/kexin type 9, acetate, and acetoacetate were increased by the intervention. Some amino acids were also altered by the intervention. The mRNA levels of LXRA and LDLR were increased, in addition to several liver X receptor α target genes and genes involved in inflammation, whereas the mRNA levels of UCP2 and PPARD were decreased in peripheral blood mononuclear cells after replacing SFAs with PUFAs. Partial least squares-discriminant analysis showed that the 30 most important variables that contributed to class separation spanned all classes of biomarkers, and was in accordance with the univariate analysis.ConclusionsApplying metabolomics in randomized controlled dietary intervention trials has the potential to extend our knowledge of the biological and molecular effects of dietary fat quality. This study was registered at clinicaltrials.gov as NCT 01679496.

Involvement of cutaneous SR-B1 in skin lipid homeostasis

BackgroundThe main functions of the skin are to protect against environmental insults and prevent water loss, which are performed by the complex lipid- and protein matrix present in the outermost layers of the epithelium. The lipidome of these outer layers is mainly composed of ceramides, fatty acids, and cholesterol, which regulates keratinocyte differentiation and skin barrier function. SR-B1 is a multifunctional scavenger receptor that is best known for facilitating uptake of cholesterol from HDL particles in the liver, but it is also expressed in the skin. ObjectiveTo determine the role of SR-B1 in keratinocyte differentiation. MethodsWe investigated the relationship between SR-B1 and keratinocyte differentiation using a physiologically relevant model, organotypic skin equivalents (SEs), wherein SR-B1 was knocked down via siRNA transfection. To assess effects of SR-B1 knockdown on keratinocyte differentiation, we performed hematoxylin/eosin staining, RT-PCR, western blotting, and immunohistochemistry. We also examined the effect of SR-B1 knockdown on lipid production by performing Oil Red O staining and thin layer chromatography. ResultsSR-B1 knockdown resulted in decreased lipid levels in SEs, specifically ceramides, and in decreased transcript levels of LDLR, PPAR-α and PPAR-γ, which are factors involved in regulating ceramide synthesis. In addition, filaggrin levels increased in SR-B1 KD tissues, but neither keratin 14 nor keratin 10 were affected. ConclusionWe conclude that one of the main functions of SR-B1 in the skin is to regulate ceramide levels and thereby maintain the barrier function of the skin, resulting in the protection of cutaneous tissues from outdoor insults.

Shuangyu Tiaozhi Granule Attenuates Hypercholesterolemia through the Reduction of Cholesterol Synthesis in Rat Fed a High Cholesterol Diet.

Shuangyu Tiaozhi Granule (STG) is composed of two kinds of Chinese medicinal herbs in dioscorea, which are used for managing cholesterol levels in patients with hypercholesterolemia in traditional Chinese medicine (TCM). However, the potential molecular mechanisms of administration of STG in hypercholesterolemia remain unknown. In this study, we investigated the effects of STG on hepatic cholesterol metabolism in high cholesterol (HC) diet-induced hypercholesterolemic rat models and simvastatin was used as a positive control. Male Sprague Dawley (SD) rats were fed general or HC diet, respectively. After 4 weeks of feeding, HC diet-induced hypercholesterolemic rats were fed HC diet, STG at 5% (w/w) or 10% (w/w) mixed in the HC diet, or HC diet combined with simvastatin gavages (4 mg·kg−1·d−1) for 4 or 8 weeks. STG treatment decreased body weight gain, liver weight ratio, serum lipids levels and hepatic lipids accumulation in rats fed a HC diet. Moreover, the effects of STG on decreasing body weight and lowering liver cholesterol levels were in dose- and time-dependent. Furthermore, STG or simvastatin treatment decreased the mRNA and protein levels of HMGCR and SREBP-2 in liver. The ACAT-2 and CYP7A1 mRNA expression were significantly decreased in HC diet supplemented with STG, while the mRNA levels of LDLR were markedly increased. STG attenuates hypercholesterolemia via inhibiting SREBP-2 signaling pathway activation and increasing hepatic uptake genes expression, providing a novel idea of TCM keeping cholesterol levels down for the clinical application.

The Cholesterol-Modulating Effect of Methanol Extract of Pigeon Pea (Cajanus cajan (L.) Millsp.) Leaves on Regulating LDLR and PCSK9 Expression in HepG2 Cells.

Pigeon pea (Cajanus cajan (L.) Millsp.) is a legume crop consumed as an indigenous vegetable in the human diet and a traditional medicinal plant with therapeutic properties. The current study highlights the cholesterol-modulating effect and underlying mechanisms of the methanol extract of Cajanus cajan L. leaves (MECC) in HepG2 cells. We found that MECC increased the LDLR expression, the cell-surface LDLR levels and the LDL uptake activity in HepG2 cells. We further demonstrated that MECC suppressed the proprotein convertase subtilisin/kexin type 9 (PCSK9) mRNA and protein expression, but not affected the expression of other cholesterol or lipid metabolism-related genes including inducible degrader of LDLR (IDOL), HMG-CoA reductase (HMGCR), fatty acid synthase (FASN), acetyl-CoA carboxylase (ACC1), and liver X receptor-α (LXR-α) in HepG2 cells. Furthermore, we demonstrated that MECC down-regulated the PCSK9 gene expression through reducing the amount of nuclear hepatocyte nuclear factor-1α (HNF-1α), a major transcriptional regulator for activation of PCSK9 promoter, but not that of nuclear sterol-responsive element binding protein-2 (SREBP-2) in HepG2 cells. Finally, we identified the cajaninstilbene acid, a main bioactive stilbene component in MECC, which significantly modulated the LDLR and PCSK9 expression in HepG2 cells. Our current data suggest that the cajaninstilbene acid may contribute to the hypocholesterolemic activity of Cajanus cajan L. leaves. Our findings support that the extract of Cajanus cajan L. leaves may serve as a cholesterol-lowering agent.