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Proprotein convertase subtilisin kexin 9 (Pcsk9) is a subtilisin serine protease with a putative role in cholesterol metabolism. Pcsk9 expression is down-regulated by dietary cholesterol, and mutations in Pcsk9 have been associated with a form of autosomal dominant hypercholesterolemia. To study the function of Pcsk9 in mice, an adenovirus constitutively expressing murine Pcsk9 (Pcsk9-Ad) was used. Pcsk9 overexpression in wild-type mice caused a 2-fold increase in plasma total cholesterol and a 5-fold increase in non-high-density lipoprotein (HDL) cholesterol, with no increase in HDL cholesterol, as compared with mice infected with a control adenovirus. Fast protein liquid chromatography analysis showed that the increase in non-HDL cholesterol was due to an increase in low-density lipoprotein (LDL) cholesterol. This effect appeared to depend on the LDL receptor (LDLR) because LDLR knockout mice infected with Pcsk9-Ad had no change in plasma cholesterol levels as compared with knockout mice infected with a control adenovirus. Furthermore, whereas overexpression of Pcsk9 had no effect on LDLR mRNA levels, there was a near absence of LDLR protein in animals overexpressing Pcsk9. These results were confirmed in vitro by the demonstration that transfection of Pcsk9 in McA-RH7777 cells caused a reduction in LDLR protein and LDL binding. In summary, these results indicate that overexpression of Pcsk9 interferes with LDLR-mediated LDL cholesterol uptake. Because Pcsk9 and LDLR are coordinately regulated by cholesterol, Pcsk9 may be involved in a novel mechanism to modulate LDLR function by an alternative pathway than classic cholesterol inhibition of sterol regulatory element binding protein-mediated transcription.

Background Microbiota and bile acids in the gastrointestinal tract profoundly alter systemic metabolic processes. In obese subjects, gradual weight loss ameliorates adipose tissue inflammation and related systemic changes. We assessed how rapid weight loss due to a very low calorie diet (VLCD) affects the fecal microbiome and fecal bile acid composition, and their interactions with the plasma metabolome and subcutaneous adipose tissue inflammation in obesity. Methods We performed a prospective cohort study of VLCD-induced weight loss of 10% in ten grades 2–3 obese postmenopausal women in a metabolic unit. Baseline and post weight loss evaluation included fasting plasma analyzed by mass spectrometry, adipose tissue transcription by RNA sequencing, stool 16S rRNA sequencing for fecal microbiota, fecal bile acids by mass spectrometry, and urinary metabolic phenotyping by 1 H-NMR spectroscopy. Outcome measures included mixed model correlations between changes in fecal microbiota and bile acid composition with changes in plasma metabolite and adipose tissue gene expression pathways. Results Alterations in the urinary metabolic phenotype following VLCD-induced weight loss were consistent with starvation ketosis, protein sparing, and disruptions to the functional status of the gut microbiota. We show that the core microbiome was preserved during VLCD-induced weight loss, but with changes in several groups of bacterial taxa with functional implications. UniFrac analysis showed overall parallel shifts in community structure, corresponding to reduced abundance of the genus Roseburia and increased Christensenellaceae;g__ (unknown genus). Imputed microbial functions showed changes in fat and carbohydrate metabolism. A significant fall in fecal total bile acid concentration and reduced deconjugation and 7-α-dihydroxylation were accompanied by significant changes in several bacterial taxa. Individual bile acids in feces correlated with amino acid, purine, and lipid metabolic pathways in plasma. Furthermore, several fecal bile acids and bacterial species correlated with altered gene expression pathways in adipose tissue. Conclusions VLCD dietary intervention in obese women changed the composition of several fecal microbial populations while preserving the core fecal microbiome. Changes in individual microbial taxa and their functions correlated with variations in the plasma metabolome, fecal bile acid composition, and adipose tissue transcriptome. Trial Registration ClinicalTrials.gov NCT01699906, 4-Oct-2012, Retrospectively registered. URL- https://clinicaltrials.gov/ct2/show/NCT01699906

Proprotein convertase subtilisin kexin 9 (PCSK9) is a member of the subtilisin serine protease family with an important role in cholesterol metabolism. PCSK9 expression is regulated by dietary cholesterol in mice and cellular sterol levels in cell culture via the sterol regulatory element binding protein transcription factors, and mutations in PCSK9 are associated with a form of autosomal dominant hypercholesterolemia. Overexpression of PCSK9 in mice leads to increased total and low-density lipoprotein (LDL) cholesterol levels because of a decrease in hepatic LDL receptor (LDLR) protein with normal mRNA levels. To study the mechanism, PCSK9 was overexpressed in human hepatoma cells, HepG2, by adenovirus. Overexpression of PCSK9 in HepG2 cells caused a decrease in whole-cell and cell-surface LDLR levels. PCSK9 overexpression had no effect on LDLR synthesis but caused a dramatic increase in the degradation of the mature LDLR and a lesser increase in the degradation of the precursor LDLR. In contrast, overexpression of a catalytically inactive mutant PCSK9 prevented the degradation of the mature LDLR; whereas increased degradation of the precursor LDLR still occurred. The PCSK9-induced degradation of the LDLR was not affected by inhibitors of the proteasome, lysosomal cysteine proteases, aspartic acid proteases, or metalloproteases. The PCSK9-induced degradation of the LDLR was shown to require transport out of the endoplasmic reticulum. These results indicate that overexpression of PCSK9 induces the degradation of the LDLR by a nonproteasomal mechanism in a post-endoplasmic reticulum compartment.

As a species the mouse is highly resistant to atherosclerosis. However, through induced mutations it has been possible to develop lines of mice that are susceptible to this disease. For example, mice that are deficient in apolipoprotein E, a ligand important in lipoprotein clearance, develop atherosclerotic lesions resembling those observed in humans. These lesions are exacerbated when the mice are fed a high-cholesterol, high-fat, Western-type diet. Other promising models are mice that are deficient in the low density lipoprotein receptor and transgenic mice that express human apolipoprotein B and transdominant mutant forms of apolipoprotein E. These models are now being used to study the pathogenesis of atherosclerotic lesions, as well as the influence of genetics, environment, hormones, and drugs on lesion development.

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Up-regulation of inflammatory responses is considered a driving force of atherosclerotic lesion development. One key regulator of inflammation is the A20 (also called TNF-α-induced protein 3 or Tnfaip3) gene, which is responsible for NF-κB termination and maps to an atherosclerosis susceptibility locus revealed by quantitative trait locus-mapping studies at mouse proximal chromosome 10. In the current study, we examined the role of A20 in atherosclerotic lesion development. At the aortic root lesion size was found to be increased in C57BL/6 (BG) apolipoprotein E-deficient (ApoE⁻/⁻) mice haploinsufficient for A20, compared with B6 ApoE⁻/⁻ controls that expressed A20 normally (60% in males and 23% in females; P < 0.001 and P < 0.05, respectively). In contrast, lesion size was found to be decreased in F₁ (B6xFVB/N) mice overexpressing A20 by virtue of containing an A20 BAC transgene compared with nontransgenic controls (30% in males, P < 0.001, and 17% in females, P = 0.02). The increase in lesions in the A20 haploinsufficient mice correlated with increased expression of proatherosclerotic NF-κB target genes, such as vascular cell adhesion molecule 1, intercellular adhesion molecule 1, and macrophage-colony-stimulating factor, and elevated plasma levels of NF-κB-driven cytokines. These findings suggest that A20 diminishes atherosclerosis by decreasing NF-κB activity, thereby modulating the proinflammatory state associated with lesion development.

Fractalkine (CX3CL1) is of particular interest in atherogenesis because it can serve as an adhesion molecule and a chemokine. Fractalkine and its receptor CX3CR1 are expressed in atherosclerotic lesions of humans and mice. However, the effect of fractalkine deficiency on atherosclerosis susceptibility is unknown. Fractalkine-deficient mice on the C57BL/6 (B6) background were bred to the atherosclerosis-sensitizing B6. ApoE-/-and B6. LDLR-/-backgrounds. Compared with controls, aortic-root lesion area was unchanged in fractalkine-deficient male and female B6. ApoE-/-mice at 16 weeks of age and males at 12 weeks of age, but it was mildly reduced (30%, P = 0.005) in females at 12 weeks of age. In contrast, lesion area at the brachiocephalic artery (BCA) was reduced dramatically by ≈85% in fractalkine-deficient females [42,251± 26,136 μ m2(n = 15) vs. 6,538± 11,320 μ m2; (n = 24), P < 0.0001] and males [36,911± 32,504 μ m2(n = 24) vs. 6,768± 8,595 μ m2(n = 14); P = 0.001] at 16 weeks of age. Fractalkine-deficient B6. ApoE-/-mice were comparable with controls in body weight, plasma cholesterol, plasma high-density lipoprotein cholesterol and white blood cell counts. On the B6. LDLR-/-background, lesion areas were reduced by 35% at the aortic root (P < 0.01) and by 50% at the BCA (P < 0.05) in fractalkine-deficient females at 16 weeks of age. Lesions in fractalkine-deficient mice on the B6. ApoE-/-and B6. LDLR-/-backgrounds were less complex and contained significantly fewer macrophages than controls. In conclusion, the major reduction of atherosclerosis in fractalkine-deficient mice appears to be at the BCA rather than the aortic root.

Non-alcoholic fatty liver disease (NAFLD) is an increasing cause of chronic liver disease that accompanies obesity and the metabolic syndrome. Excess fructose consumption can initiate or exacerbate NAFLD in part due to a consequence of impaired hepatic fructose metabolism. Preclinical data emphasized that fructose-induced altered gut microbiome, increased gut permeability, and endotoxemia play an important role in NAFLD, but human studies are sparse. The present study aimed to determine if two weeks of excess fructose consumption significantly alters gut microbiota or permeability in humans. We performed a pilot double-blind, cross-over, metabolic unit study in 10 subjects with obesity (body mass index [BMI] 30-40 mg/kg/m ). Each arm provided 75 grams of either fructose or glucose added to subjects' individual diets for 14 days, substituted isocalorically for complex carbohydrates, with a 19-day wash-out period between arms. Total fructose intake provided in the fructose arm of the study totaled a mean of 20.1% of calories. Outcome measures included fecal microbiota distribution, fecal metabolites, intestinal permeability, markers of endotoxemia, and plasma metabolites. Routine blood, uric acid, liver function, and lipid measurements were unaffected by the fructose intervention. The fecal microbiome (including ), fecal metabolites, gut permeability, indices of endotoxemia, gut damage or inflammation, and plasma metabolites were essentially unchanged by either intervention. In contrast to rodent preclinical findings, excess fructose did not cause changes in the gut microbiome, metabolome, and permeability as well as endotoxemia in humans with obesity fed fructose for 14 days in amounts known to enhance NAFLD.

Statins have adverse effects, but it's difficult to design randomized trials of alternative therapies for statin-intolerant patients. Pragmatic clinical trials, performed in the context of usual care with broad eligibility criteria, are well suited to this purpose. Statins are the most commonly prescribed drugs for lowering low-density lipoprotein (LDL) cholesterol levels. They've been amply studied in phase 3, randomized clinical trials and have been shown in primary and secondary prevention trials to reduce the risk of cardiovascular events. Statin use has increased dramatically in the past decade and will probably increase further because of lowered LDL cholesterol goals, new indications for treatment (increased C-reactive protein levels despite low or normal LDL levels), the introduction of generic versions of brand-name statins, and treatment recommendations for younger age groups. And since statin use has become the standard of care, . . .

Gene expression profiling was carried out comparing Con A elicited peritoneal macrophages from C57BL/6 and FVB/N wild-type and apolipoprotein (apo)E knockout mice. An EST, W20829, was expressed at higher levels in C57BL/6 compared with FVB/N mice. W20829 mapped to an atherosclerosis susceptibility locus on chromosome 19 revealed in an intercross between atherosclerosis-susceptible C57BL/6 and atherosclerosis-resistant FVB/N apoE knockout mice. A combination of database search and Northern analysis confirmed that W20829 corresponded to 3′-UTR of a hitherto predicted gene, named HspA12A. Blasting the National Center for Biotechnology Information database revealed a closely related homologue, HspA12B. HspA12A and -B have very close human homologues. TaqMan analysis confirmed the increased HspA12A expression (2.6-fold) in elicited peritoneal macrophages from C57BL/6 compared with FVB/N mice. TaqMan analysis also revealed increased HspA12A and HspA12B expression (87- and 6-fold, respectively) in lesional versus nonlesional portions of the thoracic aorta from C57BL/6 apoE knockout mice on a chow diet. In situ hybridization confirmed that both genes were expressed within lesions but not within nonlesional aortic tissue. Blasting of HspA12A and HspA12B against the National Center for Biotechnology Information database (NR) revealed a hit with the Conserved Domain database for Hsp70 (pfam00012.5, Hsp70). Both genes appear to contain an atypical Hsp70 ATPase domain. The Blast search also revealed that both genes were more similar to primitive eukaryote and prokaryote than mammalian Hsp70s, making these two genes distant members of the mammalian Hsp70 family. In summary, we describe two genes that code for a subfamily of Hsp70 proteins that may be involved in atherosclerosis susceptibility.