LDLR Expression Research Articles

TDP-43 mediates SREBF2-regulated gene expression required for oligodendrocyte myelination.

Cholesterol metabolism operates autonomously within the central nervous system (CNS), where the majority of cholesterol resides in myelin. We demonstrate that TDP-43, the pathological signature protein for amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), influences cholesterol metabolism in oligodendrocytes. TDP-43 binds directly to mRNA of SREBF2, the master transcription regulator for cholesterol metabolism, and multiple mRNAs encoding proteins responsible for cholesterol biosynthesis and uptake, including HMGCR, HMGCS1, and LDLR. TDP-43 depletion leads to reduced SREBF2 and LDLR expression, and cholesterol levels in vitro and in vivo. TDP-43-mediated changes in cholesterol levels can be restored by reintroducing SREBF2 or LDLR. Additionally, cholesterol supplementation rescues demyelination caused by TDP-43 deletion. Furthermore, oligodendrocytes harboring TDP-43 pathology from FTD patients show reduced HMGCR and HMGCS1, and coaggregation of LDLR and TDP-43. Collectively, our results indicate that TDP-43 plays a role in cholesterol homeostasis in oligodendrocytes, and cholesterol dysmetabolism may be implicated in TDP-43 proteinopathies-related diseases.

Lupinus angustifolius Protein Hydrolysates Reduce Abdominal Adiposity and Ameliorate Metabolic Associated Fatty Liver Disease (MAFLD) in Western Diet Fed-ApoE-/- Mice.

Metabolic-associated fatty liver disease (MAFLD) is the most important cause of liver disease worldwide. It is characterized by the accumulation of fat in the liver and is closely associated with abdominal obesity. In addition, oxidative stress and inflammation are significant features involved in MAFLD. Recently, our group demonstrated that lupin protein hydrolysates (LPHs) had lipid lowering, antioxidant, and anti-inflammatory effects. Sixty male mice fed with a Western diet were intragastrically treated with LPHs (or vehicle) for 12 weeks. Liver and adipose tissue lipid accumulation and hepatic inflammatory and oxidant status were evaluated. A significant decrease in steatosis was observed in LPHs-treated mice, which presented a decreased gene expression of CD36 and LDL-R, crucial markers in MAFLD. In addition, LPHs increased the hepatic total antioxidant capacity and reduced the hepatic inflammatory status. Moreover, LPHs-treated mice showed a significant reduction in abdominal adiposity. This is the first study to show that the supplementation with LPHs markedly ameliorates the generation of the steatotic liver caused by the intake of a Western diet and reduces abdominal obesity in ApoE−/− mice. Future clinical trials should shed light on the effects of LPHs on MAFLD.

Intraperitoneal injection of genistein affects the distribution and metabolism of cholesterol in female yellow catfish Tachysurus fulvidraco.

Genistein is an abundant phytoestrogen in soybean. This study aimed to determine the effects of genistein on cholesterol distribution and metabolism in female yellow catfish. Three hundred fish (49.2 ± 1.4g) were randomly divided into five treatments and received intraperitoneal injections as follows: (1) blank, no injection; (2) control, vehicle only; (3) E2, 17β-estradiol at 10μg·g-1 body weight; (4) low genistein doses, genistein at 10μg·g-1 body weight; (5) high genistein doses, genistein at 100μg·g-1 body weight. Both high and low genistein doses significantly reduced (p < 0.05) serum TC and LDL-C 24h after injection. Moreover, the high genistein doses significantly reduced (p < 0.05) serum HDL-C. Both high and low doses of genistein significantly increased (p < 0.05) hepatic TC. Only high genistein doses significantly increased (p < 0.05) ovary TC. In the liver, both high and low genistein doses significantly increased (p < 0.05) protein and mRNA expression of ldlr. Meanwhile, high genistein doses significantly decreased (p < 0.05) mRNA expression of hmgcr. In ovary tissue, high genistein doses significantly decreased (p < 0.05) mRNA expression of cyp11a1. These results suggested that genistein affected the cholesterol distribution in female yellow catfish. Both high and low doses of genistein reduced cholesterol content in blood and increased its content in the liver by increasing the uptake of blood cholesterol. Meanwhile, high genistein doses may inhibit hepatic cholesterol synthesis. Additionally, high genistein doses could increase cholesterol transfer from serum into the ovary and disturb cholesterol conversion to pregnenolone.

Biochanin A Regulates Cholesterol Metabolism Further Delays the Progression of Nonalcoholic Fatty Liver Disease

PurposeTo discover the possible target of biochanin A (BCA) in the lipid metabolism pathway and further explore its mechanism to nonalcoholic fatty liver disease (NAFLD).MethodsWe adopted a high-fat and high-glucose diet for 12 weeks to build the NAFLD rat model, which was then treated with different proportions of BCA for 4 weeks. General condition, body weight, Lee index, and liver index were then evaluated. Furthermore, blood lipid level and insulin resistance (IR) were detected. Moreover, hematoxylin and eosin and oil red O staining were used to observe the pathological changes in the liver. Finally, Western blotting was used to detect the protein expression levels of CYP7A1, HMGCR, LDLR, PPAR-α, PPAR-γ, and SREBP-1c in the liver.ResultsThe vital signs of rats in each group were stable. The treatment with BCA effectively reduced Lee index and liver index (F = 104.781, P < 0.05); however, the weight was not effected in each group. Additionally, BCA effectively reduced the related lipid metabolism indexes of NAFLD, such as total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), blood glucose, insulin, IR (F =12.463 (TC), 6.909 [TG], and 15.3 effected 75 [LDL], P < 0.05), and increased HDL (F = 11.580, P < 0.05). We observed that BCA could significantly improve steatosis and inflammatory cell infiltration in liver slices. Furthermore, BCA significantly increased the CYP7A1, LDLR, and PPAR-α protein expression in the liver and downregulated the HMGCR, SREBP-1c, and PPAR-γ protein expression.ConclusionBCA could delay the liver damage of NAFLD induced by a high-fat diet, regulate the blood lipid level, and improve the expression of lipid metabolism-related genes in rats.

Imperatae rhizoma-Hedyotis diffusa Willd. herbal pair alleviates nephrotic syndrome by integrating anti-inflammatory and hypolipidaemic effects

BackgroundNephrotic syndrome (NS) is a common nephropathy with a complex and diverse aetiology. Both Imperatae rhizoma and Hedyotis diffusa Willd. are herbs that are widely used as medicine and functional food. In traditional Chinese medicine theory, they are used as an herbal pair (HP) to treat inflammation-related diseases in the clinic, especially disorders of the kidney. PurposeThis study aimed to investigate the anti-inflammatory and hypolipidaemic effects of HP in an NS rat model and provide scientific data for its clinical application. MethodsAn NS model was established by two-dose injection of Sprague-Dawley rats with adriamycin. Seven groups, including the sham, model, HP treatment (0.25, 0.5 and 1.0 g/kg/d), prednisone (positive control, 5 mg/kg/d), and atorvastatin (positive control, 4 mg/kg/d) groups, were tested. The biochemical indexes of renal function and inflammatory cytokines were determined by ELISA kits and/or qPCR assays, and the crucial protein involved in the signalling pathway were subsequently tested by qPCR and/or Western blotting. Based on specific compounds identified by LC-Q-TOF-MS, network pharmacological study was carried out. ResultsThe levels of BUN, Scr, Upro, UA, Alb, TC, TG, and LDL-C were significantly elevated in model rats. HP treatment for four weeks improved the renal function and the dyslipidaemia by decreasing the levels of all parameters, except BUN and Scr. HP treatment (0.5 and 1.0 g/kg/d) upregulated the expression of PPARγ, CYP7b1, and LDLR in the liver, while it down-regulated PCSK9, showing a regulatory effect on lipid metabolism disorder. The levels of TNF-α and IL-1β in the plasma and the mRNA expression of TNF-α, IL-1β, MCP-1, and TGF-β1 in the kidney were decreased in HP groups, revealing its anti-inflammatory effect in NS rats. The HP exerted an alleviation effect on the inflammatory response through the NF-κB pathway by inhibiting the mRNA and protein expression of p50 and p65. There were 34 compounds identified or tentatively characterized in HP. In the network pharmacological study, PPARG(PPARγ), PCSK9, RELA(p65), and NF-κB1(p50) were the top 20 targets for HP, supporting the animal experimental results. Conclusion: HP exhibited protective effects on NS rats. These effects might be closely related to the inhibition of NF-κB and PCSK9-LDLR and activation of the PPARγ-CYP7B1 signalling pathways.

LDLR variants functional characterization: Contribution to variant classification

Background and aimsFamilial hypercholesterolaemia (FH) is an autosomal disorder of lipid metabolism presenting with increased cardiovascular risk. LDLR mutations are the cause of disease in 90% of the cases but functional studies have only been performed for about 15% of all LDLR variants.In the Portuguese Familial Hypercholesterolemia Study (PFHS), 142 unique LDLR alterations were identified and 44 (30%) lack functional characterization. The aim of the present work is to increase evidence for variant classification by performing functional characterization of 13 LDLR missense alterations found in Portugal and in 20 other countries. MethodsDifferent LDLR mutants were generated by site-directed mutagenesis and expressed in CHO–ldlA7 cells lacking endogenous expression of LDLR. To determine the effects of alterations on LDLR function, cell surface expression, binding and uptake of FITC-LDL were assessed by flow cytometry and Western blot. ResultsOf the 13 variants studied 7 were shown to affect LDLR function - expression, binding or uptake, with rates lower than 60%: p.(Cys184Tyr), p.(Gly207_Ser213del); p.(His211Asp); p.(Asp221Tyr); p.(Glu288Lys); p.(Gly592Glu) and p.(Asp601Val)). The remaining 6 variants do not alter the LDLR function. ConclusionsThese studies contributed to an update of these variants classification: from the 9 variants classified as variants of unknown significance, 7 have reached now a final classification and 3 variants have improved classification from likely pathogenic to pathogenic. In Portugal, an additional 55 patients received an FH definite diagnosis thanks to these studies. Since only likely pathogenic and pathogenic variants are clinically actionable, this work shows the importance of functional studies for variant classification.

β-carotene Enhances Atherosclerosis Resolution in A Reversible Murine Model of Atherosclerosis

Abstract Objectives To study the effect of β-carotene on atherosclerosis regression. Methods Wild-type mice fed a Western diet (0.3% cholesterol + 41% fat) deficient in vitamin A (WD-VAD) were injected weekly with an antisense oligonucleotide targeting the low-density lipoprotein receptor (LDLR ASO) for 16 weeks. LDLR ASO is a generous gift from Ionis Pharmaceuticals. As a result, we transiently blocked LDLR expression to facilitate the development of atherosclerotic lesions. After this period, we harvested a subset of mice (baseline group), while the remaining mice underwent atherosclerosis regression by interrupting LDLR ASO injections. To study the role of β-carotene on atherosclerosis regression, we maintained a subset of mice on WD-VAD while the remaining mice were fed WD-VAD supplemented with 50 mg/kg of β-carotene (WD-β-carotene) for 3 weeks before sacrifice. Plasma and aortic roots were collected and analyzed to study the size and composition of atherosclerotic lesions. Results Baseline mice showed a 96% (P &amp;lt; 0.0005) reduction in LDLR hepatic expression accompanied by a 3 fold (P &amp;lt; 0.0001) increased in plasma total cholesterol in comparison to both regression groups. Histological analyses of aortic roots failed to show differences in atherosclerotic lesion area between groups, although we observed a reduction in macrophage content in both regression groups that was more pronounced in WD-β-carotene-fed mice; baseline vs WD-VAD (∼30% P &amp;lt; 0.05), and baseline vs WD-β-carotene (∼42%, P &amp;lt; 0.005). Collagen content in plaques, an indicator of plaque stability in humans, showed an increase in both regression groups; baseline vs WD-VAD (∼217% P &amp;lt; 0.005), and baseline vs WD-β-carotene (∼308%, P &amp;lt; 0.0001). Conclusions β-carotene enhances atherosclerosis resolution by decreasing the inflammation and increasing plaque stability in mice. Funding Sources NIH (R01HL147252), USDA (W4002), and margin of excellence-Division of Nutritional Sciences -UIUC.

Structure–activity relationship and biological evaluation of berberine derivatives as PCSK9 down-regulating agents

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein and its deficiency markedly enhanced the survival rate of patient with cardiovascular diseases (CVDs). Forty berberine (BBR) derivatives were synthesized and evaluated for their activities on down-regulating the transcription of PCSK9 in HepG2 cells, taking BBR as the lead. Structure–activity relationship (SAR) analysis revealed that 2,3-dimethoxy moiety might be beneficial for activity. Among them, 9k displayed the most potent activity with IC50 value of 9.5 ± 0.5 μM, better than that of BBR. Also, it significantly decreased PCSK9 protein level at cellular level, as well as in the liver and serum of mice in vivo. Furthermore, 9k markedly increased LDLR expression and LDL-C clearance via down-regulating PCSK9 protein. The mechanism of action of 9k is targeting HNF1α and/or Sp1 cluster modulation upstream of PCSK9, a different one from BBR. Therefore, 9k might have the potential to be a novel PCSK9 transcriptional inhibitor for the treatment of atherosclerosis, worthy for further investigation.

LDLR inhibition promotes hepatocellular carcinoma proliferation and metastasis by elevating intracellular cholesterol synthesis through the MEK/ERK signaling pathway

ObjectiveAdaptive rewiring of cancer energy metabolism has received increasing attention. By binding with LDLs, LDLRs make most of the circulating cholesterol available for cells to utilize. However, it remains unclear how LDLR works in HCC development by affecting cholesterol metabolism. MethodsDatabase analyses and immunohistochemical staining were used to identify the clinical significance of LDLR in HCC. A transcriptome analysis was used to reveal the mechanism of LDLR aberration in HCC progression. A liver orthotopic transplantation model was used to evaluate the role of LDLR in HCC progression in vivo. ResultsDownregulation of LDLR was identified as a negative prognostic factor in human HCC. Reduced expression of LDLR in HCC cell lines impaired LDL uptake but promoted proliferation and metastasis in vitro and in vivo. Mechanistically, increasing intracellular de novo cholesterol biosynthesis was the chief contributor to malignant behaviors caused by LDLR inhibition, which could be rescued by simvastatin. Activation of the MEK/ERK pathway by LDLR downregulation partially contributed to intracellular cholesterol synthesis in HCC. ConclusionsDownregulation of LDLR may elevate intracellular cholesterol synthesis to accelerate proliferation and motility through a mechanism partially attributed to stimulation of the MEK/ERK signaling pathway. Repression of intracellular cholesterol synthesis with statins may constitute a targetable liability in the context of lower LDLR expression in HCC.

Feedback regulation of coronary artery disease susceptibility gene ADTRP and LDL receptors LDLR/CD36/LOX-1 in endothelia cell functions involved in atherosclerosis

A high level of low-density lipoprotein cholesterol (LDL) is one of the most important risk factors for coronary artery disease (CAD), the leading cause of death worldwide. However, a low concentration of LDL may be protective. Genome-wide association studies revealed that variation in ADTRP gene increased the risk of CAD. In this study, we found that a low concentration of oxidized-LDL induced the expression of ADTRP. Further analyses showed that knockdown of the expression of LDL receptor genes LDLR, CD36, or LOX-1 significantly downregulated ADTRP expression, whereas overexpression of LDLR/CD36/LOX-1 markedly increased ADTRP expression through the NF-κB pathway. Like ADTRP, LDLR, CD36 and LOX-1 were all involved in endothelial cell (EC) functions relevant to the initiation of atherosclerosis. Downregulation of LDLR/CD36/LOX-1 promoted monocyte adhesion to ECs and transendothelial migration of monocytes by increasing expression of ICAM-1, VCAM-1, E-selectin and P-selectin, decreased EC proliferation and migration, and increased EC apoptosis, thereby promoting the initiation of atherosclerosis. Opposite effects were observed with the overexpression of ADTRP and LDLR/CD36/LOX-1 in ECs. Interestingly, through the NF-κB and AKT pathways, overexpression of ADTRP significantly upregulated the expression of LDLR, CD36, and LOX-1, and knockdown of ADTRP expression significantly downregulated the expression of LDLR, CD36, and LOX-1. These data suggest that ADTRP and LDL receptors LDLR/CD36/LOX-1 positively regulate each other, and form a positive regulatory loop that regulates endothelial cell functions, thereby providing a potential protective mechanism against atherosclerosis. Our findings provide a new molecular mechanism by which deregulation of ADTRP and LDLR/CD36/LOX-1 promote the development of atherosclerosis and CAD.

Genome-scale CRISPR screening for modifiers of cellular LDL uptake.

Hypercholesterolemia is a causal and modifiable risk factor for atherosclerotic cardiovascular disease. A critical pathway regulating cholesterol homeostasis involves the receptor-mediated endocytosis of low-density lipoproteins into hepatocytes, mediated by the LDL receptor. We applied genome-scale CRISPR screening to query the genetic determinants of cellular LDL uptake in HuH7 cells cultured under either lipoprotein-rich or lipoprotein-starved conditions. Candidate LDL uptake regulators were validated through the synthesis and secondary screening of a customized library of gRNA at greater depth of coverage. This secondary screen yielded significantly improved performance relative to the primary genome-wide screen, with better discrimination of internal positive controls, no identification of negative controls, and improved concordance between screen hits at both the gene and gRNA level. We then applied our customized gRNA library to orthogonal screens that tested for the specificity of each candidate regulator for LDL versus transferrin endocytosis, the presence or absence of genetic epistasis with LDLR deletion, the impact of each perturbation on LDLR expression and trafficking, and the generalizability of LDL uptake modifiers across multiple cell types. These findings identified several previously unrecognized genes with putative roles in LDL uptake and suggest mechanisms for their functional interaction with LDLR.

Impact of bone marrow ATP-binding cassette transporter A1 deficiency on atherogenesis is independent of the presence of the low-density lipoprotein receptor

Background and aimsThere is extensive evidence from bone marrow transplantation studies that hematopoietic ATP binding cassette A1 (Abca1) is atheroprotective in low-density lipoprotein receptor (Ldlr) deficient mice. In contrast, studies using lysosyme M promoter-driven deletion of Abca1 in Ldlr deficient mice failed to show similar effects. It was hypothesized that the discrepancy between these studies might be due to the presence of Ldlr in bone marrow-derived cells in the transplantation model. In this study, we aim to determine the contribution of Ldlr to the atheroprotective effect of hematopoietic Abca1 in the murine bone marrow transplantation model. MethodsWild-type, Ldlr−/−, Abca1−/−, and Abca1−/−Ldlr−/− bone marrow was transplanted into hypercholesterolemic Ldlr−/− mice. ResultsBone marrow Lldr deficiency did not influence the effects of Abca1 on macrophage cholesterol efflux, foam cell formation, monocytosis or plasma cholesterol. Ldlr deficiency did reduce circulating and peritoneal lymphocyte counts, albeit only in animals lacking Abca1 in bone marrow-derived cells. Importantly, the effects of Abca1 deficiency on atherosclerosis susceptibility were unaltered by the presence or absence of Ldlr. Bone marrow Ldlr deficiency did lead to marginally but consistently decreased atherosclerosis, regardless of Abca1 deficiency. Thus, Ldlr expression on bone marrow-derived cells does, to a minimal extent, influence atherosclerotic lesion development, albeit independent of Abca1. ConclusionsThis study provides novel insight into the relative impact of Ldlr and Abca1 in bone marrow-derived cells on macrophage foam cell formation and atherosclerosis development in vivo. We have shown that Ldlr and Abca1 differentially and independently influence atherosclerosis development in a murine bone marrow transplantation model of atherosclerosis.

Exosome-based Ldlr gene therapy for familial hypercholesterolemia in a mouse model.

Familial hypercholesterolemia (FH), with high LDL (low-density lipoprotein) cholesterol levels, is due to inherited mutations in genes, such as low-density lipoprotein receptor (LDLR). Development of therapeutic strategies for FH, which causes atherosclerosis and cardiovascular disease, is urgently needed.Methods: Mice with low-density lipoprotein receptor (Ldlr) deletion (Ldlr-/- mice) were used as an FH model. Ldlr mRNA was encapsulated into exosomes by forced expression of Ldlr in the donor AML12 (alpha mouse liver) cells, and the resultant exosomes were denoted as ExoLdlr. In vivo distribution of exosomes was analyzed by fluorescence labeling and imaging. The delivery efficiency of Ldlr mRNA was analyzed by qPCR and Western blotting. Therapeutic effects of ExoLdlr were examined in Ldlr-/- mice by blood lipids and Oil Red O staining.Results: The encapsulated mRNA was stable and could be translated into functional protein in the recipient cells. Following tail vein injection, exosomes were mainly delivered into the liver, producing abundant LDLR protein, resembling the endogenous expression profile in the wild-type mouse. Compared with control exosomes, ExoLdlr treatment significantly decreased lipid deposition in the liver and lowered the serum LDL-cholesterol level. Significantly, the number and size of atherosclerotic plaques and inflammation were reduced in the ExoLdlr-treated mice.Conclusions: We have shown that exosome-mediated Ldlr mRNA delivery effectively restored receptor expression, treating the disorders in the Ldlr-/- mouse. Our study provided a new therapeutic approach for the treatment of FH patients and managing atherosclerosis.

Mechanism of long non‑coding RNA metastasis‑associated lung adenocarcinoma transcript 1 in lipidmetabolism and inflammation in heart failure.

Heart failure (HF) is a serious threat to human health. Long noncoding RNAs (lncRNAs) are critical regulators of HF. The aim of the study was to investigate the molecular mechanism of MALAT1 in HF rats. MALAT1 expression was detected in serum of normal volunteers and HF patients, HF rats and isoproterenol (ISO)-induced H9C2 cells, and its diagnostic value was evaluated in HF patients. Indexes related to cardiac functions and hemodynamics, myocardial injury, lipid metabolism, lipid oxidation, and inflammation were detected. Moreover, the downstream mechanism of MALAT1 was predicted and verified and in vivo experiments were further performed in ISO-induced H9C2 cells to verify the effects of MALAT1 in HF. MALAT1 was highly expressed in serum of HF patients, HF rats and ISO-induced H9C2 cells and was valuable in predicting HF. Inhibition of MALAT1 increased cardiac function and anti-inflammation and alleviated myocardial injury, lipid metabolism, lipid oxidation and apoptosis rates. Inhibition of MALAT1 reduced H9C2 cell injury. MALAT1 competitively bound to microRNA (miR)-532-3p to upregulate LDLR protein. Inhibition of miR-532-3p weakened the protective effect of downregulated MALAT1 against H9C2 cell injury. We concluded that MALAT1 upregulated LDLR expression by competitively binding to miR-532-3p, thereby increasing pathological injury in HF.

Small Molecule Phenotypic Screen Identifies Novel Regulators of LDLR Expression.

Alzheimer's Disease (AD) is a progressive neurodegenerative disease and the most common cause of dementia. The current treatment options for AD are limited to ameliorating cognitive decline temporarily and not reversing or preventing the progression of dementia. Hence, more effective therapeutic strategies are needed to combat this devastating disease. The low-density lipoprotein receptor has been shown to modulate the neuronal metabolism of cholesterol and apolipoprotein E, a major genetic risk factor for AD. LDLR overexpression in mice has been shown to increase amyloid-β clearance and reduce amyloid deposition. We conducted a phenotypic screen to identify novel signaling pathways and targets that regulate LDLR expression in glial cells using an annotated compound library of approximately 29 000 compounds. The screen identified novel targets such as polo like kinase 1 (PLK1), activin receptor like kinase 5 (ALK5), and serotonin transporter (SERT). We used genetic, chemical biology and pathway analysis to confirm the target hypothesis. This work highlights that phenotypic screening is a promising strategy to identify novel mechanisms and targets for therapeutic intervention of complex neurodegenerative disorders.

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.

Proteostasis Regulation in the Endoplasmic Reticulum: An Emerging Theme in the Molecular Pathology and Therapeutic Management of Familial Hypercholesterolemia.

Familial hypercholesterolemia (FH) is an autosomal genetic disease characterized by high serum low-density lipoprotein (LDL) content leading to premature coronary artery disease. The main genetic and molecular causes of FH are mutations in low-density lipoprotein receptor gene (LDLR) resulting in the non-clearance of LDL from the blood by hepatocytes and consequently the formation of plaques. LDLR is synthesized and glycosylated in the endoplasmic reticulum (ER) and then transported to the plasma membrane via Golgi. It is estimated that more than 50% of reported FH-causing mutations in LDLR result in misfolded proteins that are transport-defective and hence retained in ER. ER accumulation of misfolded proteins causes ER-stress and activates unfolded protein response (UPR). UPR aids protein folding, blocks further protein synthesis, and eliminates misfolded proteins via ER-associated degradation (ERAD) to alleviate ER stress. Various studies demonstrated that ER-retained LDLR mutants are subjected to ERAD. Interestingly, chemical chaperones and genetic or pharmacological inhibition of ERAD have been reported to rescue the transport defective mutant LDLR alleles from ERAD and restore their ER-Golgi transport resulting in the expression of functional plasma membrane LDLR. This suggests the possibility of pharmacological modulation of proteostasis in the ER as a therapeutic strategy for FH. In this review, we picture a detailed analysis of UPR and the ERAD processes activated by ER-retained LDLR mutants associated with FH. In addition, we discuss and critically evaluate the potential role of chemical chaperones and ERAD modulators in the therapeutic management of FH.

INFLAMMATORY MACROPHAGES DERIVED EXOSOMAL MIR-222 PROMOTED THECA CELL STEROIDOGENESIS BY MODULATING MYLIP EXPRESSION

To explore the role of M1 macrophages (M1M) in the linkage between low-grade inflammation and ovarian dysfunction in obesity. In vitro experimental laboratory study. Raw264.7 macrophage was treated with lipopolysaccharide (LPS) to establish a model of M1M. Exosome (Exo) secreted by macrophage was isolated by ultracentrifugation and identified by transmission electron microscopy (TEM), western blot, and flow nanoanalyzer. The primary murine ovary theca cell (TC) was co-incubated with PKH67 fluorescence-labeled Exo to detect whether Exo secreted by macrophage could be uptaken by TC. The expression of CYP11A1 and CYP17A1 were analyzed by qPCR and western blot. Medium testosterone and progesterone level were analyzed. High-throughput sequencing was performed to analyze the expression of exosomal miRNA (miRNA-seq). In order to select key differential exosomal miRNA, the GSE97652 (species: mouse) was downloaded from the GEO DataBases. The data set contains 7 replications in both experimental group and control group. The experimental group is obese mouse adipose tissue macrophage (ATM)-derived Exo, and the control group is control mouse ATM -derived Exo. The overlap between miRNA-seq and GSE97652 differentially expressed miRNA was analyzed according to the Fold change > 2, p < 0.01 standard, which determined miR-222 and MYLIP for subsequent experiments. Next, gain-of-function experiments were performed to examine their role in TC steroidogenesis function with the involvement of the cholesterol metabolism pathway. TEM, Flow NanoAnalyzer and western blot analysis confirmed the successful extraction of Exo. After co-cultured TC with PKH67-labeled Exo, green fluorescence could be detected in TC which demonstrated that TC was able to ingest Exo secreted by the macrophage. M1M increased the expression of CYP11A1 and the production of progesterone in TC which is dependent upon M1-Exo. The result of miRNA-seq showed that there were 30 upregulated miRNA and 112 down-regulated miRNA in M1M derived Exo compared with control Exo according to the standard of Fold change > 2(p < 0.05). A total of two differentially expressed miRNA, namely miR-222 and miR-155, were selected from the venn diagram of miRNA-seq and GSE97652. QPCR verified that miR-222 in both M1M derived Exo and M1M derived Exo treated TC were significantly upregulated. Subsequently, we predicted the target gene of miR-222 through five different databases. MYLIP, with the highest target score was selected, which has been identified as a key factor promoting LDLR degradation. M1M derived Exo was verified to display a high expression level of miR-222, and M1M derived Exo mediated TC steroidogenesis depended miR-222. The overexpression of miR-222 resulted in the down-regulation of MYLIP expression and up-regulation of LDLR expression, followed by increased cholesterol influx, CYP11A1 overexpression and progesterone overproduction. M1M derived Exo can be ingested by TC. M1M promote TC CYP11A1 overexpression and progesterone overproduction through Exo by delivering miR-222 into TC.

Familial hypercholesterolemia: A single-nucleotide variant (SNV) in mosaic at the low density lipoprotein receptor (LDLR)

Background and aimsFamilial hypercholesterolemia is most frequently caused by genetic variants in the LDLR gene. Most of LDLR pathogenic variants are missense, followed by splicing and deletion/insertions variants. Mosaicism is a genetic condition in which an individual shows more than one clone of cells with different genotypes. The objective of this article was the molecular characterization of a patient with hypercholesterolemia. Methods and resultsGenetic analysis of DNA from peripheral blood and saliva was performed by NGS, Sanger sequencing and pyrosequencing technologies. NGS analysis detected the pathogenic variant LDLR:c.1951G >T:p.(Asp651Tyr) in 9%–12% of reads. The presence of the variant was confirmed by pyrosequencing analysis. The variant found was functional characterized using an in vitro model (CHO-ldlA7 cells). Activity and expression of cell surface LDLR were measured by flow cytometry. Colocalization LDLR-Dil-LDL was detected by immunofluorescence. The LDLR activity showed 80% uptake, 50% binding and 53% expression of cell surface LDLR regarding wild type. ConclusionsHerein, we report the first case of a mosaic single nucleotide variant affecting the LDLR gene in a patient with familial hypercholesterolemia. As it has been described for other pathologies, mosaicism could be underestimated in FH and its detection will improve with the introduction of NGS technologies in the diagnostic routine.

Zearalenone disrupts the placental function of rats: A possible mechanism causing intrauterine growth restriction

Zearalenone is an estrogenic mycotoxin produced by a variety of Fusarium fungi. There is evidence that exposure to zearalenone can cause intrauterine growth restriction, but little is known about the mechanism in the rat placenta caused by zearalenone. From gestational day 14–21, female Sprague Dawley rats (60 days old) were gavaged with zearalenone (0, 2.5, 5, 10, and 20 mg/kg/day body weight). Zearalenone dose-dependently reduced serum LH and FSH levels of dams at ≥ 5 mg/kg. RNA-seq and qPCR showed that zearalenone significantly down-regulated Slc38a1 expression at 2.5 mg/kg, Echs1 and Pc at 10 mg/kg, as well as Slc1a5, Cd36, Ldlr, Hadhb, and Cyp17a1 expression at a dose of 20 mg/kg, while it up-regulated the expression of Notch signal (Dvl1 and Jag 1). After zearalenone treatment, their proteins showed a similar trend. Zearalenone reduced the phosphorylation of AKT1, ERK1/2, and mTOR at 5 mg/kg or higher and 4EBP1 at 5 mg/kg. Zearalenone also increased BECLIN1, LC3B, and p62 levels and elevated BAX/BCL2 and CASP3/PROCASP3 ratios. In conclusion, zearalenone disrupts placental function such as reduction of nutrient transport and lipid metabolism possibly via AKT1/ERK1/2/mTOR-mediated autophagy and apoptosis.