apoE mRNA Research Articles

Effects of Microglial Cytokines on Alzheimer's Disease-Related Phenomena.

Recent association studies indicate several genes highly expressed by microglia influence Alzheimer's disease (AD) risk, which suggests microglial function contributes to this disease. Here, we evaluated how one component of microglial function, cytokine release, affects AD-related phenomena. First, we used a 3-hour lipopolysaccharide (LPS) treatment to activate mouse BV2 microglial cells. Next, we removed the LPS-containing medium, added LPS-free medium, and after 6 hours collected the medium conditioned by the activated BV2 microglial cells. We then exposed human neuronal SH-SY5Y cells to the conditioned medium for 24 hours. At the end of the 24-hour exposure, we assessed amyloid-β protein precursor (AβPP), tau, apolipoprotein E (ApoE), and lipid status. The amount of AβPP was unaffected, although a slight decrease in soluble AβPPα suggested a subtle reduction in AβPP non-amyloidogenic processing occurred. Tau mRNA increased, but total and phosphorylated tau levels were unchanged. ApoE mRNA increased, while ApoE protein levels were lower. Per cell lipid droplet number decreased and lipid oxidation increased. These results show cytokine release by activated microglial cells can influence specific AD-relevant physiologies and pathologies.

Quercetin decreases the triglyceride content through the PPAR signalling pathway in primary hepatocytes of broiler chickens

This study was conducted to investigate the effect of quercetin on the triglyceride (TG) content through the peroxisome proliferator-activated receptor α (PPARα) signalling pathway in broiler hepatocytes. The trial included the following five groups: control, DMSO control, 5 mg/L quercetin, 10 mg/L quercetin and 20 mg/L quercetin. Compared with the control group, the PPARα protein content was significantly increased at 24 h in the groups that received quercetin at 5, 10 and 20 mg/L (p < 0.05), whereas the PPARα mRNA expression was significantly increased at 24, 48 and 72 h (p < 0.05). Compared with the control, the content of the long-chain acyl-CoA synthetase (ACSL), apolipoprotein A1 (ApoA1), liver fatty acid–binding protein (FABP) and TG in the 5-, 10- and 20-mg/L quercetin group was significantly increased at 24, 48 and 72 h (p < 0.05). Moreover, the L-FABP and ApoA1 mRNA expression levels were significantly increased at 24, 48 and 72 h (p < 0.05). Compared with the control, the ApoC3 protein content in the 5-mg/L quercetin group was significantly decreased at 72 h (p < 0.05). Furthermore, the very-low-density lipoprotein (VLDL) contents in the 5- and 20-mg/L quercetin groups were significantly decreased at 72 h (p < 0.05). In conclusion, quercetin enhanced lipid transportation and peroxidase β-oxidation regulated by PPARα and promoted lipid decomposition regulated by PPARα, but this did not occur in a dose-dependent or time-dependent manner. Therefore, quercetin decreased the TG content in broiler hepatocytes by promoting PPARα-regulated lipid decomposition, thereby reducing lipid deposition in broilers.

Silencing of PPARαBb mRNA in brown trout primary hepatocytes: effects on molecular and morphological targets under the influence of an estrogen and a PPARα agonist.

The crosstalk between peroxisome proliferator-activated receptor α (PPARα) and estrogenic pathways are shared from fish to humans. Salmonid fish had an additional genome duplication, and two PPARα isoforms (PPARαBa and PPARαBb) were previously identified. Since a negative regulation between estrogen signaling and PPARα was described, a post-transcriptional gene silencing for PPARαBb was designed in primary brown trout hepatocytes. The aims of the study were to: (i) decipher the effects of PPARαBb knock-down on peroxisome morphology and on mRNA expression of potential target genes, and (ii) to assess the cross-interferences caused by an estrogenic compound (17α-ethinylestradiol - EE2) and a PPARα agonist (Wy-14,643 - Wy) using the established knock-down model. A knock-down efficiency of 70% was achieved for PPARαBb and its silencing significantly reduced the volume density of peroxisomes, but did not alter mRNA levels of the studied genes. Exposure to Wy did not change peroxisome morphology or mRNA expression, but under silencing conditions Wy rescued the volume density of peroxisomes to control levels, and increased acyl-coenzyme A oxidase 1-3l (Acox1-3l) mRNA. Exposure to EE2 caused a reduction of peroxisome volume density, but under silencing conditions this effect was abolished and ApoA1 mRNA level was diminished. The morphological alterations of peroxisomes by WY and EE2 demonstrated that obtained results are PPARαBb dependent, and suggest the regulation of unknown downstream targets of PPARαBb. In summary, PPARαBb is involved in the control of peroxisome size and/or number, which opens future opportunities to explore its regulation and molecular targets.

Typha angustifolia extract reduces diet-induced hyperlipidemia in rats

ObjectiveTo observe the lipid-lowering effect of Typha angustifolia extract (TAE) in Sprague Dawley rats. MethodsThe rats were randomly divided into six groups, including the hypercholesterolemic diet (HYD) group. Ad libitum food was given to the animals for 60 d to establish dyslipidemia models. The rats were received an ig administration of 1 mL medicine daily. After 120 d the animals were sacrificed by cervical dislocation; Blood was collected to measure total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein-cholesterol (HDL-C), nitric oxide (NO), and endothelin (ET) plasma concentration; Livers were collected to measure ApoE mRNA and protein expression; Morphologic changes of aorta ventralis tissue were also observed. ResultsCompared with HYD group, TAE had the ability of reducing TC, TG, LDL-C, NO, and ET (P < 0.01), thereby increasing ApoE mRNA and protein expression of the liver (P < 0.01). ConclusionThese results suggested that TAE was capable of effectively decreasing the circulating lipids levels and enhancing the protective effects of artery.

Large‐Scale Screening of Natural Products Transactivating Peroxisome Proliferator‐Activated Receptor α Identifies 9S‐Hydroxy‐10E,12Z,15Z‐Octadecatrienoic Acid and Cymarin as Potential Compounds Capable of Increasing Apolipoprotein A‐I Transcription in Human Liver Cells

Increasing apolipoprotein A-I (apoA-I), the predominant protein of high-density lipoprotein (HDL) particles, has favorable effects on atherogenic risk factors. Here, we investigated the effects of peroxisome proliferator-activated receptor α (PPARα) transactivating compounds on apoA-I transcription in HepG2 cells. A transient PPARα agonist transactivation assay was used to screen 2500 natural compounds. To analyze the effects on apoA-I transcription, human hepatocellular liver carcinoma (HepG2) were exposed to 0.1, 1, and 10 μg/mL of the natural PPARα transactivators. ApoA-I mRNA expression was determined by quantitative polymerase chain reaction. Extensive dose-response experiments were performed using compounds that increased apoA-I transcription by minimally 20%. Kelch-like ECH-associated protein 1 (KEAP) and carnitine palmitoyltransferase 1 alpha (CPT1α) expression were used respectively to confirm Bromodomain-containing protein 4 inhibition or PPARα activation. Twenty-eight natural compounds increased PPARα transactivation by at least twofold. Despite the increased CPT1α expression seen after the addition of most PPARα activating compounds, CPT1α expression and PPARα transactivation did not correlate. Addition of 0.05 μg/mL 9S-hydroxy-10E,12Z,15Z-octadecatrienoic acid (9(S)-HOTrE) increased apoA-I mRNA expression by 35%, whereas 10-25 μg/mL of cymarin increased apoA-I transcription by 37%. However, combining cymarin and 9(S)-HOTrE did not result in a synergistic effect, in contrast this combination even decreased apoA-I transcription. ApoA-I transcription involves multiple regulatory players, and PPARα transactivation alone is not sufficient. A search for natural compounds resembling the molecular structure of 9(S)-HOTrE or cymarin could aid to find additional components that increase apoA-I transcription.

Apolipoprotein E Affects In Vitro Axonal Growth and Regeneration via the MAPK Signaling Pathway

Following central nervous system injury in mammals, failed axonal regeneration is closely related to dysneuria. Previous studies have shown that the obvious effects of apolipoprotein E (ApoE) on traumatic brain injury (TBI) were associated with an axonal mechanism. However, little information on the actions of ApoE and its isoforms on axonal regeneration following TBI was provided. In our study, the cerebral cortices of ApoE-deficient (ApoE-/-) and wild-type (ApoE +/+ ) mice were cultured in vitro, and an axonal transection model was established. Interventions included the conditioned medium of astrocytes, human recombinant ApoE2/3/4 isoforms and inhibitors of the JNK/ERK/p38 pathway. Axonal growth and regeneration were evaluated by measuring the maximum distance and area of the axons. The expression levels of β-tubulin III, MAP2, ApoE, p-JNK, p-ERK and p-p38 were detected by immunofluorescence and western blotting. The results showed that ApoE mRNA and protein were expressed in intact axons and regenerated axons. Axonal growth and regeneration were attenuated in ApoE-/- mice but recovered by exogenous ApoE. Human recombinant ApoE3 positively influenced axonal growth and regeneration; these effects were mediated by the JNK/ERK/p38 pathway. These results suggest ApoE and its isoforms may have influenced axonal growth and regeneration via the MAPK signaling pathway in vitro.

Increased apolipoprotein M induced by lack of scavenger receptor BI is not activated via HDL-mediated cholesterol uptake in hepatocytes

BackgroundScavenger receptor BI (SR-BI) is a classic high-density lipoprotein (HDL) receptor, which mediates selective lipid uptake from HDL cholesterol esters (HDL-C). Apolipoprotein M (ApoM), as a component of HDL particles, could influence preβ-HDL formation and cholesterol efflux. The aim of this study was to determine whether SR-BI deficiency influenced the expression of ApoM.MethodsBlood samples and liver tissues were collected from SR-BI gene knockout mice, and serum lipid parameters, including total cholesterol (TC), triglyceride (TG), high and low-density lipoprotein cholesterol (HDL-C and LDL-C) and ApoM were measured. Hepatic ApoM and ApoAI mRNA levels were also determined. In addition, BLT-1, an inhibitor of SR-BI, was added to HepG2 cells cultured with cholesterol and HDL, under serum or serum-free conditions. The mRNA and protein expression levels of ApoM were detected by RT-PCR and western blot.ResultsWe found that increased serum ApoM protein levels corresponded with high hepatic ApoM mRNA levels in both male and female SR-BI−/− mice. Besides, serum TC and HDL-C were also significantly increased. Treatment of HepG2 hepatoma cells with SR-BI specific inhibitor, BLT-1, could up-regulate ApoM expression in serum-containing medium but not in serum-free medium, even in the presence of HDL-C and cholesterol.ConclusionsResults suggested that SR-BI deficiency promoted ApoM expression, but the increased ApoM might be independent from HDL-mediated cholesterol uptake in hepatocytes.

NEURONAL APOE MODULATES A SPORADIC ALZHEIMER’S DISEASE PHENOTYPE IN PATIENT-DERIVED INDUCED NEURONS

The APOE-ε4 allele is the strongest genetic risk factor for sporadic Alzheimer's disease (sAD) and may be enriched in other clinically relevant populations. While APOE is predominantly expressed by astrocytes in the central nervous system, neuronal expression of APOE is of increasing interest in age-related cognitive impairment, neurological injury, and neurodegeneration. The human and mouse APOE promoter sequences are dissimilar, and contain distinct regions that control expression in a species specific, cell-type specific manner. APOE mRNA and protein is detectable in primary human neuronal cultures in a manner suggestive of de novo protein synthesis. However, neuronal expression of APOE has not been linked to neurotoxicity in a human system. We reprogrammed induced pluripotent stem cells (iPSCs) from three unrelated sAD patients with the ε3/ε4 genotype, used CRISPR/Cas9 gene editing to generate ε3/ε3 isogenic lines, and differentiated them into pure cultures of glutamatergic neurons with no detectable glia. APOE is synthesized and secreted by human induced neurons. Genetic correction of ε4 to ε3 reduces tau phosphorylation and phosphoactivation of a specific kinase. Moreover, ε4 is associated with altered amyloid secretion, and exacerbates the neurotoxic effects of a calcium ionophore. APOE is expressed in human glia-free neuronal culture system, and APOE isoform alters sAD-associated molecular pathways. More detailed mechanistic studies are required to better understand this phenomenon. However, the net effect is that endogenous expression of APOE-ε4 weakens neurons and contributes to the sAD cellular phenotype independently of, but likely cooperatively with, glial APOE.

LPCAT3 deficiency in hematopoietic cells alters cholesterol and phospholipid homeostasis and promotes atherosclerosis

Background and aimsLPCAT3 plays a major role in phospholipid metabolism in the liver and intestine. However, the impact of LPCAT3 on hematopoietic cell and macrophage functions has yet to be described. Our aim was to understand the functions of LPCAT3 in macrophages and to investigate whether LPCAT3 deficiency in hematopoietic cells may affect atherosclerosis development. MethodsMice with constitutive Lpcat3 deficiency (Lpcat3−/−) were generated. We used fetal hematopoietic liver cells to generate WT and Lpcat3−/− macrophages in vitro and to perform hematopoietic cell transplantation in recipient Ldlr−/− mice. ResultsLpcat3-deficient macrophages displayed major reductions in the arachidonate content of phosphatidylcholines, phosphatidylethanolamines and, unexpectedly, plasmalogens. These changes were associated with altered cholesterol homeostasis, including an increase in the ratio of free to esterified cholesterol and a reduction in cholesterol efflux in Lpcat3−/− macrophages. This correlated with the inhibition of some LXR-regulated pathways, related to altered cellular availability of the arachidonic acid. Indeed, LPCAT3 deficiency was associated with decreased Abca1, Abcg1 and ApoE mRNA levels in fetal liver cells derived macrophages. In vivo, these changes translated into a significant increase in atherosclerotic lesions in Ldlr−/− mice with hematopoietic LPCAT3 deficiency. ConclusionsThis study identifies LPCAT3 as a key factor in the control of phospholipid homeostasis and arachidonate availability in myeloid cells and underlines a new role for LPCAT3 in plasmalogen metabolism. Moreover, our work strengthens the link between phospholipid and sterol metabolism in hematopoietic cells, with significant consequences on nuclear receptor-regulated pathways and atherosclerosis development.

The microRNA-1908 up-regulation in the peripheral blood cells impairs amyloid clearance by targeting ApoE.

To give a new insight into the mechanism of ApoE dysregulation and microRNA-1908 in Alzheimer's disease (AD). Plasma ApoE levels were measured in 20 AD patients and 20 healthy controls. THP-1 was maintained in RPMI1640 with 10% fetal bovine serum. Quantitative real-time polymerase chain reaction was performed to detect 13-microRNA and ApoE mRNA in cultured cell lines. Enzyme-linked immunosorbent assay was used to measure human ApoE in the plasma or culture medium of cell lines and also used to quantify the human Aβ42 in the culture medium of cell lines. We found plasma ApoE level reduced in AD patients (2.28 vs 3.78μg/mL, P<.001), and microRNA-1908 was up-regulated in AD patients and was negatively associated with plasma ApoE (r=-0.32, P=.012). In human macrophage cell line THP-1 and astrocytoma cell line U87, microRNA-1908 could inhibit the mRNA and protein levels of ApoE by targeting its 3'untranslated region. Consistently, microRNA-1908 inhibits the ApoE-mediated Aβ clearance. Our study provides new insight into the mechanism of ApoE dysregulation in AD patients, and microRNA-1908 might be a therapeutic target for AD treatment.

Apolipoprotein E4 impairs spontaneous blood brain barrier repair following traumatic brain injury

BackgroundTraumatic Brain Injury (TBI) is a major cause of disability and mortality, to which there is currently no comprehensive treatment. Blood Brain Barrier (BBB) dysfunction is well documented in human TBI patients, yet the molecular mechanisms that underlie this neurovascular unit (NVU) pathology remains unclear. The apolipoprotein-E (apoE) protein has been implicated in controlling BBB integrity in an isoform dependent manner, via suppression of Cyclophilin A (CypA)–Matrix metallopeptidase-9 (MMP-9) signaling cascades, however the contribution of this pathway in TBI-induced BBB permeability is not fully investigated.MethodsWe exposed C57Bl/6 mice to controlled cortical impact and assessed NVU and BBB permeability responses up to 21 days post-injury. We pharmacologically probed the role of the CypA-MMP-9 pathway in BBB permeability after TBI using Cyclosporin A (CsA, 20 mg/kg). Finally, as the apoE4 protein is known to be functionally deficient compared to the apoE3 protein, we used humanized APOE mice as a clinically relevant model to study the role of apoE on BBB injury and repair after TBI.ResultsIn C57Bl/6 mice there was an inverse relationship between soluble apoE and BBB permeability, such that damaged BBB stabilizes as apoE levels increase in the days following TBI. TBI mice displayed acute pericyte loss, increased MMP-9 production and activity, and reduced tight-junction expression. Treatment with the CypA antagonist CsA in C57Bl/6 mice attenuates MMP-9 responses and enhances BBB repair after injury, demonstrating that MMP-9 plays an important role in the timing of spontaneous BBB repair after TBI. We also show that apoe mRNA is present in both astrocytes and pericytes after TBI.We report that APOE3 and APOE4 mice have similar acute BBB responses to TBI, but APOE3 mice display faster spontaneous BBB repair than APOE4 mice. Isolated microvessel analysis reveals delayed pericyte repopulation, augmented and sustained MMP-9 expression at the NVU, and impaired stabilization of Zonula Occludens-1, Occludin and Claudin-5 expression at tight junctions in APOE4 mice after TBI compared to APOE3 mice.ConclusionsThese data confirm apoE as an important modulator of spontaneous BBB stabilization following TBI, and highlights the APOE4 allele as a risk factor for poor outcome after TBI.

Tumor necrosis factor α stimulates endogenous apolipoprotein A-I expression and secretion by human monocytes and macrophages: role of MAP-kinases, NF-κB, and nuclear receptors PPARα and LXRs.

Apolipoprotein A-I (ApoA-I) is the main structural and functional protein component of high-density lipoprotein. ApoA-I has been shown to regulate lipid metabolism and inflammation in macrophages. Recently, we found the moderate expression of endogenous apoA-I in human monocytes and macrophages and showed that pro-inflammatory cytokine tumor necrosis factor α (TNFα) increases apoA-I mRNA and stimulates ApoA-I protein secretion by human monocytes and macrophages. Here, we present data about molecular mechanisms responsible for the TNFα-mediated activation of apoA-I gene in human monocytes and macrophages. This activation depends on JNK and MEK1/2 signaling pathways in human monocytes, whereas inhibition of NFκB, JNK, or p38 blocks an increase of apoA-I gene expression in the macrophages treated with TNFα. Nuclear receptor PPARα is a ligand-dependent regulator of apoA-I gene, whereas LXRs stimulate apoA-I mRNA transcription and ApoA-I protein synthesis and secretion by macrophages. Treatment of human macrophages with PPARα or LXR synthetic ligands as well as knock-down of LXRα, and LXRβ by siRNAs interfered with the TNFα-mediated activation of apoA-I gene in human monocytes and macrophages. At the same time, TNFα differently regulated the levels of PPARα, LXRα, and LXRβ binding to the apoA-I gene promoter in THP-1 cells. Obtained results suggest a novel tissue-specific mechanism of the TNFα-mediated regulation of apoA-I gene in monocytes and macrophages and show that endogenous ApoA-I might be positively regulated in macrophage during inflammation.

A pleiotropic effect of the APOE gene: association of APOE polymorphisms with multibacillary leprosy in Han Chinese from Southwest China.

Patients with leprosy have a very low risk of Alzheimer disease (AD) and β-amyloid (Aβ) deposition is significantly lower in the brain tissue of elderly patients with leprosy compared with age-matched controls. Apolipoprotein E (ApoE) plays a critical role in lipid metabolic pathways and in the brain, facilitating the proteolytic clearance of Aβ. We hypothesized that APOE confers risk of leprosy as lipid metabolism is involved in Mycobacterium leprae infection. To investigate the potential genetic associations between APOE and leprosy in two independent Chinese case-control cohorts from the Yuxi and Wenshan prefectures, Yunnan Province of Southwest China. Five APOE single-nucleotide polymorphisms (SNPs) were analysed in 1110 individuals (527 patients and 583 controls) from the Yuxi prefecture using a SNaPshot assay. Genetic variations in the entire APOE exons were screened in 1788 individuals (798 patients and 990 controls) from the Wenshan prefecture using next-generation sequencing technology. The AD-associated SNPs rs405509 and rs439401 increased the risk of leprosy per se and multibacillary leprosy (P<0·005), but the APOE-ε4 allele did not. The SNPs rs405509 and rs439401 were cis expression quantitative trait loci (eQTL) for APOE expression in human skin. Differential APOE mRNA expression was observed in skin lesions of patients with type I reaction leprosy and those with multibacillary leprosy. APOE and related lipid genes are involved in an interaction network with leprosy susceptibility genes. The APOE gene is associated with leprosy, most likely by regulating lipid-metabolism-related genes.

Apolipoprotein A1 distribution pattern in the human Achilles tendon.

Metabolic factors such as cholesterol appear to play an important role in the development of Achilles tendinopathy. There is, however, no morphologic proof explaining the link between high cholesterol and tendinopathy. As apolipoprotein A1 (Apo-A1) is essential for reverse cholesterol transport, it may be related to cholesterol overload in tendon. Nothing is known about Apo-A1 expression in tendon tissue. We examined the distribution of Apo-A1 protein in biopsies from normal and tendinopathy-affected human Achilles tendons, and APOA1 mRNA production from cultured human hamstring tenocytes. Specific immunoreactions for Apo-A1 were detected. The tenocytes showed specific Apo-A1 immunoreactions. These reactions were usually distinct in the tendinopathy specimens. While the tendinopathy specimens often showed granular/small deposit reactions, the slender tenocytes of control specimens did not show this pattern. The magnitude of Apo-A1 immunoreactivity was especially marked in the tendinopathy specimens, as there is a high number of tenocytes. Reactions were also seen in the walls of blood vessels located within the tendon tissue proper of both the normal and tendinopathy tendons and within the peritendinous/fatty tissue of the tendinopathy tendons. The reactions were predominantly in the form of deposit reactions within the smooth muscle layer of the vessel walls. Cultured hamstring tenocytes produced APOA1 mRNA. We demonstrated the presence of Apo-A1 in human tendon tissue. This suggests there may be a link between Achilles tendinopathy and cholesterol metabolism. We hypothesize that Apo-A1 may be important for tenocyte and blood vessel function within tendons.

Silencing steroid receptor coactivator-1 in the nucleus of the solitary tract reduces estrogenic effects on feeding and apolipoprotein A-IV expression

We previously found that 17β-estradiol (E2) stimulates apolipoprotein A-IV (apoA-IV) gene expression in the nucleus of the solitary tract (NTS) of lean ovariectomized (OVX) rodents. Here we report that in the NTS of high-fat diet-induced obese (DIO) rats, the apoA-IV mRNA level is significantly reduced and that the estrogenic effects on apoA-IV gene expression and food intake are impaired. E2 regulates apoA-IV gene expression through its nuclear receptor α (ERα), which requires co-activators, such as steroid receptor coactivator-1 (SRC-1), to facilitate the transcription of targeted genes. Interestingly, SRC-1 gene expression is significantly reduced in DIO OVX rats. SRC-1 is colocalized with apoA-IV in the cells of the NTS and E2 treatment enhances the recruitment of ERα and SRC-1 to the estrogen response element at the apoA-V promoter, implying the participation of SRC-1 in E2's stimulatory effect on apoA-IV gene expression. Using small hairpin RNA (shRNA), which was validated in cultured neuronal cells, we found that SRC-1 gene knockdown specifically in the NTS significantly diminished E2's anorectic action, leading to increased food intake and body weight. More importantly, the stimulatory effect of E2 on apoA-IV gene expression in the NTS was significantly attenuated in SRC-1 knockdown rats. These results collectively demonstrate the critical roles of NTS SRC-1 in mediating E2's actions on food intake and apoA-IV gene expression and suggest that reduced levels of endogenous SRC-1 and apoA-IV expression are responsible for the impaired E2's anorectic action in obese females.

The acute effects on duodenal gene expression in healthy men following consumption of a low-fat meal enriched with theobromine or fat

Increasing apoA-I synthesis may improve HDL functionality and lower CVD risk. As theobromine and fat increase fasting apoA-I concentrations, and the intestine is involved in apoA-I production, the acute effects of both were studied on duodenal gene transcription to better understand underlying mechanisms. In this crossover study, 8 healthy men received once a low fat (LF) meal, a LF meal plus theobromine (850 mg), or a high fat (HF) meal. Five hours after meal intake duodenal biopsies were taken for microarray analysis. Theobromine and HF consumption did not change duodenal apoA-I expression. Theobromine did not change gene expression related to lipid and cholesterol metabolism, whereas those related to glycogen/glucose breakdown were downregulated. HF consumption increased gene expression related to lipid and cholesterol uptake and transport, and to glucose storage, while it decreased those related to glucose uptake. Furthermore, genes related to inflammation were upregulated, but inflammation markers in plasma were not changed. In healthy men, acute theobromine and fat consumption did not change duodenal apoA-I mRNA, but inhibited expression of genes related to glucose metabolism. Furthermore, HF intake activated in the duodenum expression of genes related to lipid and cholesterol metabolism and to inflammation.

Molecular mechanisms of tributyltin-induced alterations in cholesterol homeostasis and steroidogenesis in hamster testis: In vivo and in vitro studies.

Tributyltins (TBT) are ubiquitous and persistent environmental contaminants that disturb normal endocrine function including gonadal function in humans and marine organisms. TBT was administered through oral route to male Syrian hamsters at daily doses of 50, 100, and 150 ppm/kg for 65 days. Changes in testis morphology, immunohistochemistry of iNOS, 3β-HSD, and 17β-HSD, cholesterol transport receptor, nuclear receptors, and transcription factors were analyzed. TBT treatment affected each of these parameters to significant levels in a dose-dependent manner compared to vehicle treated control. Real-time PCR and protein analyses revealed that expression levels of ApoE and LDL-R mRNA were up-regulated in the testis of TBT-treated animals while the expression levels of SR-B1, LXR, PPARs α, β, and γ, SCAP, SREBP 1 and 2, 3β-HSD, 17β-HSD, CYP17A1, and P450SCC were down-regulated. Leydig cells were isolated and separated adopting percoll gradient centrifugation under aseptic condition. The viability of Leydig cell was affected by TBT treatment in a dose- and time-dependent manner. Further, the mechanism of action of TBT was ascertained by siRNA transfection of ApoE, which was upregulated, and SREBP, which was down-regulated. These observations led us to infer that exposure to TBT hinders intracellular cholesterol transport resulting in abnormal sex steroid biosynthesis and alteration of steroidogenic enzyme activities. Finally, we could recognize ApoE and SREBP as the major factors regulating genes that control cholesterol biosynthesis and steroidogenesis that ultimately inhibit the synthesis of testosterone. Therefore, ApoE is one of the important molecular targets that can be intercepted in context of male infertility/male contraception.

Diallyl disulphide inhibits apolipoprotein(a) expression in HepG2 cells through the MEK1-ERK1/2-ELK-1 pathway

BackgroundLipoprotein(a) [LP(a)] is implicated as a common and independent risk factor for cardiovascular diseases. The therapeutic options currently available for reducing plasma LP(a) concentrations are limited. Diallyl disulphide (DADS), the main component of garlic, regulates lipid metabolism in hepatocytes and adipocytes through ERK1/2 signalling. This study aimed to assess the effect of DADS on apolipoprotein(a) [apo(a)] in HepG2 cells. We also determined the effects of DADS on apo(a) expression and secretion in HepG2 cells as well as the underlying mechanisms.MethodsWe examined the role of DADS on apo(a) expression in HepG2 cells by treating cell with different concentrations of DADS (10, 20, 40 and 80 μg/mL) for 24 h or treating cells with 40 μg/mL DADS for 0, 6, 12, 24 and 48 h. Then we used quantitative real-time PCR to analysis apo(a) mRNA levels, used Western blot to analysis apo(a) protein levels and used enzyme-linked immunosorbent assay to test apo(a) secreted levels. To farther determined the role of DADS, we applied Transfection of small interfering RNA to knockdown ELK-1levels and applied PD98059, a specific inhibitor of ERK1/2, to block ERK1/2 signal.ResultsThe results show DADS inhibited apo(a) at both the mRNA and protein levels in HepG2 cells in a dose-dependent manner. DADS-mediated inhibition of apoa(a) expression in HepG2 cells was attenuated when the cells were cultured in medium containing PD98059 (ERK1/2 inhibitor) or were transfected with siRNAs against MEK1 or ELK-1. Overexpression of apo(a) yielded similar results.ConclusionsThis study reveals that DADS can downregulate apo(a) expression in a dose-dependent manner via the MEK-ERK12-ELK-1 pathway.

Metformin improves nonalcoholic fatty liver disease in obese mice via down-regulation of apolipoprotein A5 as part of the AMPK/LXRα signaling pathway.

Apolipoprotein A5 (apoA5) has been implicated in the formation of hepatocyte lipid droplets, a histological hallmark of non-alcoholic fatty liver disease (NAFLD). Recent evidence demonstrated that liver X receptor α (LXRα), a transcription factor involved in down-regulation of APOA5 mRNA, is activated by AMP-activated protein kinase (AMPK) that contributes to metformin-related antihyperglycemic effects. In this study we investigated the role of apoA5 and AMPK/LXRα signaling pathway in metformin-related improvement of NAFLD. Leptin-deficient (ob/ob) obese mice with NAFLD were treated with metformin, and signaling pathways were compared with non-metformin treated mice. Additionally, we determined cellular apoA5 and triglyceride (TG) levels in mouse hepatocytes in vitro and the effects of metformin, with or without an AMPK inhibitor or LXRα siRNA, on these levels. We found that metformin dose-dependently ameliorated hepatosteatosis and liver dysfunction in ob/ob mice, with a significant reduction in hepatic apoA5 expression and TG level. Metformin also dose-dependently increased phosphorylation of hepatic AMPK and LXRα in ob/ob mice. Similarly, metformin decreased apoA5 expression and TG level in mouse hepatocytes, with increased phosphorylation of cellular AMPK and LXRα. Addition of AMPK inhibitor or siRNA knockdown of LXRα significantly attenuated metformin-induced down-regulation of cellular apoA5 expression and TG level. AMPK inhibitor also significantly inhibited metformin-induced LXRα phosphorylation in these hepatocytes. Therefore, our findings indicate that metformin improves obesity-related NAFLD via inhibition of hepatic apoA5 synthesis as part of the AMPK/LXRα signaling pathway.

Effect of multiple neonatal sevoflurane exposures on hippocampal apolipoprotein E levels and learning and memory abilities

Sevoflurane anesthesia is widely used in pediatric patients. In this study, we investigated whether early multiple exposures to sevoflurane induced cognitive dysfunction by altering the hippocampal expression of ApoE later in development. Sprague-Dawley rats were exposed to 2.6% sevoflurane at postnatal day 7 (P7), P14, and P21 for 2h. The ability of learning and memory was assessed using the Morris water maze at P37 and P97. The hippocampal volume was measured by magnetic resonance imaging (MRI) at P37 and P97. The hippocampal expression of ApoE was assessed by immunohistochemical analyses and real-time polymerase chain reaction (PCR). Behavioral testing revealed that the ability of learning and memory in the sevoflurane-exposed rats was decreased compared with the control animals; however, there was no significant difference (P>0.05). The MRI results showed a significant decrease in the left hippocampal volume, left maximum hippocampal length, and right maximum hippocampal length in the sevoflurane young group compared with the control young group (P<0.05). The brain volume, left maximum hippocampal length, right hippocampal volume, and maximum brain length were significantly lower in the sevoflurane adult group than in the control adult group (P<0.05). In young animals, the ApoE expression in the hippocampal CA1 and CA3 regions and the ApoE mRNA level were significantly higher compared with the control group (P<0.05), but not in the dentate gyrus region (P>0.05). Among the adult animals, there was no significant difference between the groups in any parameter tested (P>0.05). Multiple exposures to sevoflurane during the neonatal period decreased the volume of the hippocampus and increased the hippocampal expression of ApoE. The differential expression level of ApoE in different hippocampal subdivisions suggested that the expression of ApoE was regionally specific and reversible.