Hepatic SREBP-2 and cholesterol biosynthesis are regulated by FoxO3 and Sirt6

Cholesterol homeostasis is crucial for cellular function and organismal health. The key regulator for the cholesterol biosynthesis is sterol-regulatory element binding protein (SREBP)-2. The biochemical process and physiological function of SREBP-2 have been well characterized; however, it is not clear how this gene is epigenetically regulated. Here we have identified sirtuin (Sirt)6 as a critical factor for Srebp2 gene regulation. Hepatic deficiency of Sirt6 in mice leads to elevated cholesterol levels. On the mechanistic level, Sirt6 is recruited by forkhead box O (FoxO)3 to the Srebp2 gene promoter where Sirt6 deacetylates histone H3 at lysines 9 and 56, thereby promoting a repressive chromatin state. Remarkably, Sirt6 or FoxO3 overexpression improves hypercholesterolemia in diet-induced or genetically obese mice. In summary, our data suggest an important role of hepatic Sirt6 and FoxO3 in the regulation of cholesterol homeostasis.     

PCSK9 is expressed in pancreatic δ-cells and does not alter insulin secretion

PCSK9 (Proprotein Convertase Subtilisin Kexin type 9) is a proprotein convertase that plays a key role in cholesterol homeostasis by decreasing hepatic low-density lipoprotein receptor (LDLR) protein expression. Here, we investigated the expression and the function of PCSK9 in pancreatic islets. Immunohistochemistry analysis showed that PCSK9 co-localized specifically with somatostatin in human pancreatic δ-cells, with no expression in α- and β-cells. PCSK9 seems not to be secreted by mouse isolated islets maintained in culture. Pcsk9-deficiency led to a 200% increase in LDLR protein content in mouse isolated islets, mainly in β-cells. Conversely, incubation of islets with recombinant PCSK9 almost abolished LDLR expression. However, Pcsk9-deficiency did not alter cholesterol content nor glucose-stimulated insulin secretion in mouse islets. Finally, invivo glucose tolerance was similar in Pcsk9^+/+ and Pcsk9^−/− mice under basal conditions and following streptozotocin treatment. These results suggest, at least in mice, that PCSK9 does not alter insulin secretion.     

Progression of Chronic Liver Inflammation and Fibrosis Driven by Activation of c-JUN Signaling in Sirt6 Mutant Mice

The human body has a remarkable ability to regulate inflammation, a biophysical response triggered by virus infection and tissue damage. Sirt6 is critical for metabolism and lifespan; however, its role in inflammation is unknown. Here we show that Sirt6-null (Sirt6^−/−) mice developed chronic liver inflammation starting at ∼2 months of age, and all animals were affected by 7–8 months of age. Deletion of Sirt6 in T cells or myeloid-derived cells was sufficient to induce liver inflammation and fibrosis, albeit to a lesser degree than that in the global Sirt6^−/− mice, suggesting that Sirt6 deficiency in the immune cells is the cause. Consistently, macrophages derived from the bone marrow of Sirt6^−/− mice showed increased MCP-1, IL-6, and TNFα expression levels and were hypersensitive to LPS stimulation. Mechanistically, SIRT6 interacts with c-JUN and deacetylates histone H3 lysine 9 (H3K9) at the promoter of proinflammatory genes whose expression involves the c-JUN signaling pathway. Sirt6-deficient macrophages displayed hyperacetylation of H3K9 and increased occupancy of c-JUN in the promoter of these genes, leading to their elevated expression. These data suggest that Sirt6 plays an anti-inflammatory role in mice by inhibiting c-JUN-dependent expression of proinflammatory genes.     

Anacetrapib reduces (V)LDL cholesterol by inhibition of CETP activity and reduction of plasma PCSK9

Recently, we showed in APOE*3-Leiden cholesteryl ester transfer protein (E3L.CETP) mice that anacetrapib attenuated atherosclerosis development by reducing (V)LDL cholesterol [(V)LDL-C] rather than by raising HDL cholesterol. Here, we investigated the mechanism by which anacetrapib reduces (V)LDL-C and whether this effect was dependent on the inhibition of CETP. E3L.CETP mice were fed a Western-type diet alone or supplemented with anacetrapib (30 mg/kg body weight per day). Microarray analyses of livers revealed downregulation of the cholesterol biosynthesis pathway (P < 0.001) and predicted downregulation of pathways controlled by sterol regulatory element-binding proteins 1 and 2 (z-scores −2.56 and −2.90, respectively; both P < 0.001). These data suggest increased supply of cholesterol to the liver. We found that hepatic proprotein convertase subtilisin/kexin type 9 (Pcsk9) expression was decreased (−28%, P < 0.01), accompanied by decreased plasma PCSK9 levels (−47%, P < 0.001) and increased hepatic LDL receptor (LDLr) content (+64%, P < 0.01). Consistent with this, anacetrapib increased the clearance and hepatic uptake (+25%, P < 0.001) of [¹⁴C]cholesteryl oleate-labeled VLDL-mimicking particles. In E3L mice that do not express CETP, anacetrapib still decreased (V)LDL-C and plasma PCSK9 levels, indicating that these effects were independent of CETP inhibition. We conclude that anacetrapib reduces (V)LDL-C by two mechanisms: 1) inhibition of CETP activity, resulting in remodeled VLDL particles that are more susceptible to hepatic uptake; and 2) a CETP-independent reduction of plasma PCSK9 levels that has the potential to increase LDLr-mediated hepatic remnant clearance.     

Improved efficacy for ezetimibe and rosuvastatin by attenuating the induction of PCSK9

Reducing circulating LDL-cholesterol (LDL-c) reduces the risk of cardiovascular disease in people with hypercholesterolemia. Current approaches to reduce circulating LDL-c include statins, which inhibit cholesterol synthesis, and ezetimibe, which blocks cholesterol absorption. Both elevate serum PCSK9 protein levels in patients, which could attenuate their efficacy by reducing the amount of cholesterol cleared from circulation. To determine whether PCSK9 inhibition could enhance LDL-c lowering of both statins and ezetimibe, we utilized small interfering RNAs (siRNAs) to knock down Pcsk9, together with ezetimibe, rosuvastatin, and an ezetimibe/rosuvastatin combination in a mouse model with a human-like lipid profile. We found that ezetimibe, rosuvastatin, and ezetimibe/rosuvastatin combined lower serum cholesterol but induce the expression of Pcsk9 as well as the Srebp-2 hepatic cholesterol biosynthesis pathway. Pcsk9 knockdown in combination with either treatment led to greater reductions in serum non-HDL with a near-uniform reduction of all LDL-c subfractions. In addition to reducing serum cholesterol, the combined rosuvastatin/ezetimibe/Pcsk9 siRNA treatment exhibited a significant reduction in serum APOB protein and triglyceride levels. Taken together, these data provide evidence that PCSK9 inhibitors, in combination with current therapies, have the potential to achieve greater reductions in both serum cholesterol and triglycerides.     

Antisense inhibition of proprotein convertase subtilisin/kexin type 9 reduces serum LDL in hyperlipidemic mice

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a member of a family of proteases that is thought to promote the degradation of the low density lipoprotein receptor (LDLR) through an as yet undefined mechanism. We developed second generation antisense oligonucleotide (ASO) inhibitors targeting murine PCSK9 to determine their potential as lipid-lowering agents. Administration of a PCSK9 ASO to high fat-fed mice for 6 weeks reduced total cholesterol and LDL by 53% and 38%, respectively. Moreover, inhibition of PCSK9 expression resulted in a 2-fold increase in hepatic LDLR protein levels. This phenotype closely resembles that reported previously in Pcsk9-deficient mice. The absence of cholesterol lowering in Ldlr-deficient mice effectively demonstrated a critical role for this receptor in mediating the lipid-lowering effects of PCSK9 inhibition. Antisense inhibition of PCSK9 is an attractive and novel therapeutic approach for treating hypercholesterolemia in human.     

PCSK9 reduces the protein levels of the LDL receptor in mouse brain during development and after ischemic stroke

Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a major role in cholesterol homeostasis through enhanced degradation of the LDL receptor (LDLR) in liver. As novel inhibitors/silencers of PCSK9 are now being tested in clinical trials to treat hypercholesterolemia, it is crucial to define the physiological consequences of the lack of PCSK9 in various organs. LDLR regulation by PCSK9 has not been extensively described during mouse brain development and injury. Herein, we show that PCSK9 and LDLR are co-expressed in mouse brain during development and at adulthood. Although the protein levels of LDLR and apolipoprotein E (apoE) in the adult brain of Pcsk9(-/-) mice are similar to those of wild-type (WT) mice, LDLR levels increased and were accompanied by a reduction of apoE levels during development. This suggests that the upregulation of LDLR protein levels in Pcsk9(-/-) mice enhances apoE degradation. Upon ischemic stroke, PCSK9 was expressed in the dentate gyrus between 24 h and 72 h following brain reperfusion. Although mouse behavior and lesion volume were similar, LDLR protein levels dropped ～2-fold less in the Pcsk9(-/-)-lesioned hippocampus, without affecting apoE levels and neurogenesis. Thus, PCSK9 downregulates LDLR levels during brain development and following transient ischemic stroke in adult mice.     

PCSK9: an enigmatic protease

Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a critical role in cholesterol metabolism by controlling the levels of low density lipoprotein (LDL) particles that circulate in the bloodstream. Several gain-of-function and loss-of-function mutations in the PCSK9 gene, that occur naturally, have been identified and linked to hypercholesterolemia and hypocholesterolemia, respectively. PCSK9 expression has been shown to be regulated by sterol regulatory element binding proteins (SREBPs) and statins similar to other genes involved in cholesterol homeostasis. The most critical finding concerning PCSK9 is that this protease is able to influence the number of LDL receptor molecules expressed on the cell surface. Studies have demonstrated that PCSK9 acts mainly by enhancing degradation of LDL receptor protein in the liver. Inactivation of PCSK9 in mice reduces plasma cholesterol levels primarily by increasing hepatic expression of LDL receptor protein and thereby accelerating clearance of circulating LDL cholesterol. The objective of this review is to summarize the current information related to the regulation and function of PCSK9 and to identify gaps in our present knowledge.     

Suppressor of Cytokine Signaling-3 (SOCS-3) Induces Proprotein Convertase Subtilisin Kexin Type 9 (PCSK9) Expression in Hepatic HepG2 Cell Line

The suppressor of cytokine signaling (SOCS) proteins are negative regulators of the JAK/STAT pathway activated by proinflammatory cytokines, including the tumor necrosis factor-α (TNF-α). SOCS3 is also implicated in hypertriglyceridemia associated to insulin resistance. Proprotein convertase subtilisin kexin type 9 (PCSK9) levels are frequently found to be positively correlated to insulin resistance and plasma very low density lipoprotein (VLDL) triglycerides concentrations. The present study aimed to investigate the possible role of TNF-α and JAK/STAT pathway on de novo lipogenesis and PCSK9 expression in HepG2 cells. TNF-α induced both SOCS3 and PCSK9 in a concentration-dependent manner. This effect was inhibited by transfection with siRNA anti-STAT3, suggesting the involvement of the JAK/STAT pathway. Retroviral overexpression of SOCS3 in HepG2 cells (HepG2^SOCS3) strongly inhibited STAT3 phosphorylation and induced PCSK9 mRNA and protein, with no effect on its promoter activity and mRNA stability. Consistently, siRNA anti-SOCS3 reduced PCSK9 mRNA levels, whereas an opposite effect was observed with siRNA anti-STAT3. In addition, HepG2^SOCS3 express higher mRNA levels of key enzymes involved in the de novo lipogenesis, such as fattyacid synthase, stearoyl-CoA desaturase (SCD)-1, and apoB. These responses were associated with a significant increase of SCD-1 protein, activation of sterol regulatory element-binding protein-1c (SREBP-1), accumulation of cellular triglycerides, and secretion of apoB. HepG2^SOCS3 show lower phosphorylation levels of insulin receptor substrate 1 (IRS-1) Tyr⁸⁹⁶ and Akt Ser⁴⁷³ in response to insulin. Finally, insulin stimulation produced an additive effect with SOCS3 overexpression, further inducing PCSK9, SREBP-1, fatty acid synthase, and apoB mRNA. In conclusion, our data candidate PCSK9 as a gene involved in lipid metabolism regulated by proinflammatory cytokine TNF-α in a SOCS3-dependent manner.     

Modulation of Sirt1 by resveratrol and nicotinamide alters proliferation and differentiation of pig preadipocytes

Sirt1, a NAD⁺-dependent histone deacetylase, may regulate senescence, metabolism, and apoptosis. In this study, primary pig preadipocytes were cultured in DMEM/F12 medium containing 10% fetal bovine serum (FBS) with or without reagents affecting Sirt1 activity. The adipocyte differentiation process was visualized by light microscopy after Oil red O staining. Proliferation and differentiation of preadipocytes was measured using methylthiazolyldiphenyl-tetrazolium bromide (MTT) and Oil red O extraction. Expression of Sirt1, FoxO1, and adipocyte specific genes was detected with semi-quantitive RT-PCR. The results showed that Sirt1 mRNA was widely expressed in various pig tissues from different developmental stages. Sirt1 mRNA was expressed throughout the entire differentiation process of pig preadipocytes. Resveratrol significantly increased Sirt1 mRNA expression, but decreased the expression of FoxO1 and adipocyte marker gene PPARγ2. Resveratrol significantly inhibited pig preadipocyte proliferation and differentiation. Nicotinamide decreased the expression of Sirt1 mRNA, but increased the expression of FoxO1 and adipocyte specific genes. Nicotinamide greatly stimulated the proliferation and differentiation of pig preadipocytes. In conclusion, these results indicate that Sirt1 may modulate the proliferation and differentiation of pig preadipocytes. Sirt1 may down-regulate pig preadipocytes proliferation and differentiation through repression of adipocyte genes or FoxO1.     

PCSK9 inhibition fails to alter hepatic LDLR,circulating cholesterol,and atherosclerosis in the absence of ApoE

LDL cholesterol (LDL-C) contributes to coronary heart disease. Proprotein convertase subtilisin/kexin type 9 (PCSK9) increases LDL-C by inhibiting LDL-C clearance. The therapeutic potential for PCSK9 inhibitors is highlighted by the fact that PCSK9 loss-of-function carriers exhibit 15–30% lower circulating LDL-C and a disproportionately lower risk (47–88%) of experiencing a cardiovascular event. Here, we utilized pcsk9^−/− mice and an anti-PCSK9 antibody to study the role of the LDL receptor (LDLR) and ApoE in PCSK9-mediated regulation of plasma cholesterol and atherosclerotic lesion development. We found that circulating cholesterol and atherosclerotic lesions were minimally modified in pcsk9^−/− mice on either an LDLR- or ApoE-deficient background. Acute administration of an anti-PCSK9 antibody did not reduce circulating cholesterol in an ApoE-deficient background, but did reduce circulating cholesterol (−45%) and TGs (−36%) in APOE*3Leiden.cholesteryl ester transfer protein (CETP) mice, which contain mouse ApoE, human mutant APOE3*Leiden, and a functional LDLR. Chronic anti-PCSK9 antibody treatment in APOE*3Leiden.CETP mice resulted in a significant reduction in atherosclerotic lesion area (−91%) and reduced lesion complexity. Taken together, these results indicate that both LDLR and ApoE are required for PCSK9 inhibitor-mediated reductions in atherosclerosis, as both are needed to increase hepatic LDLR expression.     

FoxO1对下丘脑摄食中枢的影响研究进展

下丘脑是人体的摄食中枢,它通过抑制食欲的阿黑皮素原(POMC)神经元和促进食欲的神经肽相关蛋白(AgRP)神经元调节摄食及能量代谢。叉头转录因子O亚族1(FoxO1)是胰岛素信号通路和瘦素信号通路中重要的调节蛋白,FoxO1的生理作用是促进下丘脑Agrp基因表达、抑制Pomc基因表达,抑制瘦素信号通路的转录激活因子3(STAT3)蛋白对Pomc基因转录的促进作用,从而促进食欲。瘦素和胰岛素均可激活经典的IRS/PI(3)K/Akt信号通路,使FoxO1磷酸化失去活性,抑制食欲。此外,沉默信息调节因子Sirt1也可以通过去乙酰化,影响FoxO1的转录活性。本文综述了胰岛素、瘦素、Sirt1通过FoxO1调节下丘脑摄食中枢的作用机制。     

Pax6 Directly Down-Regulates Pcsk1n Expression Thereby Regulating PC1/3 Dependent Proinsulin Processing

Background

Heterozygous paired box6 (Pax6) mutations lead to abnormal glucose metabolism in mice older than 6 months as well as in human beings. Our previous study found that Pax6 deficiency caused down-expression of prohormone convertase 1/3 (Pcsk1), resulting in defective proinsulin processing. As a protein cleaving enzyme, in addition to its expression, the activity of PC1/3 is closely related to its function. We therefore hypothesize that Pax6 mutation alters the activity of PC1/3, which affects proinsulin processing.

Methodology/Principal Findings

Using quantitative RT-PCR, western blot and enzyme assay, we found that PC1/3 C-terminal cleavage and its activity were compromised in Pax6 R266Stop mutant mice, and the expression of Pcsk1n, a potent inhibitor of PC1/3, was elevated by Pax6 deficiency in the mutant mice and MIN6 cells. We confirmed the effect of proSAAS, the protein encoded by Pcsk1n, on PC1/3 C-terminal cleavage and its activity by Pcsk1n RNAi in MIN6 cells. Furthermore, by luciferase-reporter analysis, chromatin immunoprecipitation, and electrophoretic mobility shift assay, we revealed that Pax6 bound to Pcsk1n promoter and directly down-regulated its expression. Finally, by co-transfecting Pax6 siRNA with Pcsk1n siRNA, we showed that Pax6 knock-down inhibited proinsulin processing and that this effect could be rescued by proSAAS down-regulation. These findings confirm that Pax6 regulates proinsulin processing partially through proSAAS-mediated PC1/3 processing and activity.

Conclusions/Significance

Collectively, the above experiments demonstrate that Pax6 can directly down-regulate Pcsk1n expression, which negatively affects PC1/3 C-terminal cleavage and activity and subsequently participates in proinsulin processing. We identified proSAAS as a novel down-regulated target of Pax6 in the regulation of glucose metabolism. This study also provides a complete molecular mechanism for the Pax6 deficiency-caused diabetes.     

A new method for measurement of total plasma PCSK9: clinical applications

The proprotein convertase subtilisin kexin-9 (PCSK9) circulates in plasma as mature and furin-cleaved forms. A polyclonal antibody against human PCSK9 was used to develop an ELISA that measures total plasma PCSK9 rather than only the mature form. A cross-sectional study evaluated plasma levels in normal (n = 254) and hypercholesterolemic (n = 200) subjects treated or untreated with statins or statin plus ezetimibe. In controls, mean plasma PCSK9 (89.5 ± 31.9 ng/ml) correlated positively with age, total cholesterol, LDL-cholesterol (LDL-C), triglycerides, and fasting glucose. Sequencing PCSK9 from individuals at the extremes of the normal PCSK9 distribution identified a new loss-of-function R434W variant associated with lower levels of circulating PCSK9 and LDL-C. In hypercholesterolemic subjects, PCSK9 levels were higher than in controls (99.3 ± 31.7 ng/ml, P < 0.04) and increased in proportion to the statin dose, combined or not with ezetimibe. In treated patients (n = 139), those with familial hypercholesterolemia (FH; due to LDL receptor gene mutations) had higher PCSK9 values than non-FH (147.01 ± 42.5 vs. 127.2 ± 40.8 ng/ml, P < 0.005), but LDL-C reduction correlated positively with achieved plasma PCSK9 levels to a similar extent in both subsets (r = 0.316, P < 0.02 in FH and r = 0.275, P < 0.009 in non-FH). The detection of circulating PCSK9 in both FH and non-FH subjects means that this assay could be used to monitor response to therapy in a wide range of patients.