Sirt6 deletion in bone marrow-derived cells increases atherosclerosis - Central role of macrophage scavenger receptor 1

Aims: Sirtuin 6 (Sirt6) is a NAD(+)-dependent deacetylase that plays a key role in DNA repair, inflammation and lipid regulation. Sirt6-null mice show severe metabolic defects and accelerated aging. Macrophage-foam cell formation via scavenger receptors is a key step in atherogenesis. We determined the effects of bone marrow-restricted Sirt6 deletion on foam cell formation and atherogenesis using a mouse model. Methods and results: Sirt6 deletion in bone marrow-derived cells increased aortic plaques, lipid content and macrophage numbers in recipient Apoe(-/-) mice fed a high-cholesterol diet for 12 weeks (n = 12-14, p < .001). In RAW macrophages, Sirt6 overexpression reduced oxidized low-density lipoprotein (oxLDL) uptake, Sirt6 knockdown enhanced it and increased mRNA and protein levels of macrophage scavenger receptor 1 (Msr1), whereas levels of other oxLDL uptake and efflux transporters remained unchanged. Similarly, in human primary macrophages, Sirt6 knockdown increased MSR1 protein levels and oxLDL uptake. Double knockdown of Sirt6 and Msr1 abolished the increase in oxLDL uptake observed upon Sirt6 single knockdown. FACS analyses of macrophages from aortic plaques of Sirt6-deficient bone marrow-transplanted mice showed increased MSR1 protein expression. Double knockdown of Sirt6 and the transcription factor c-Myc in RAW cells abolished the increase in Msr1 mRNA and protein levels; c-Myc overexpression increased Msr1 mRNA and protein levels. Conclusions: Loss of Sirt6 in bone marrow-derived cells is proatherogenic; hereby macrophages play an important role given a c-Myc-dependent increase in MSR1 protein expression and an enhanced oxLDL uptake in human and murine macrophages. These findings assign endogenous SIRT6 in macrophages an important atheroprotective role.

The Sirt1 activator SRT3025 provides atheroprotection in Apoe(-/-) mice by reducing hepatic Pcsk9 secretion and enhancing Ldlr expression

Tasneem Arsiwala, Johan Auwerx, Kristina Schoonjans, Matthias Alexander Sokrates Stein

The deacetylase sirtuin 1 (Sirt1) exerts beneficial effects on lipid metabolism, but its roles in plasma LDL-cholesterol regulation and atherosclerosis are controversial. Thus, we applied the pharmacological Sirt1 activator SRT3025 in a mouse model of atherosclerosis and in hepatocyte culture. Apolipoprotein E-deficient (Apoe(-/-)) mice were fed a high-cholesterol diet (1.25% w/w) supplemented with SRT3025 (3.18 g kg(-1) diet) for 12 weeks. In vitro, the drug activated wild-type Sirt1 protein, but not the activation-resistant Sirt1 mutant; in vivo, it increased deacetylation of hepatic p65 and skeletal muscle Foxo1. SRT3025 treatment decreased plasma levels of LDL-cholesterol and total cholesterol and reduced atherosclerosis. Drug treatment did not change mRNA expression of hepatic LDL receptor (Ldlr) and proprotein convertase subtilisin/kexin type 9 (Pcsk9), but increased their protein expression indicating post-translational effects. Consistent with hepatocyte Ldlr and Pcsk9 accumulation, we found reduced plasma levels of Pcsk9 after pharmacological Sirt1 activation. In vitro administration of SRT3025 to cultured AML12 hepatocytes attenuated Pcsk9 secretion and its binding to Ldlr, thereby reducing Pcsk9-mediated Ldlr degradation and increasing Ldlr expression and LDL uptake. Co-administration of exogenous Pcsk9 with SRT3025 blunted these effects. Sirt1 activation with SRT3025 in Ldlr(-/-) mice reduced neither plasma Pcsk9, nor LDL-cholesterol levels, nor atherosclerosis. We identify reduction in Pcsk9 secretion as a novel effect of Sirt1 activity and uncover Ldlr as a prerequisite for Sirt1-mediated atheroprotection in mice. Pharmacological activation of Sirt1 appears promising to be tested in patients for its effects on plasma Pcsk9, LDL-cholesterol, and atherosclerosis.

Oxford University Press2015

Functional analysis of Bicc1 and novel interacting proteins in renal tubule cell metabolism and polycystic kidneys

Lucía Carolina Leal Esteban

Genetic disorders known as ciliopathies develop polycystic kidneys, including autosomal dominant and autosomal recessive polycystic kidney diseases (ADPKD and ARPKD). Several signaling pathways including the cAMP/PKA pathway are implicated in driving cystic growth. However, the mechanisms underlying cysts formation is still elusive. Renal cysts also arise in patients and in mouse models with mutations in the RNA-binding protein Bicaudal-C (Bicc1). Bicc1 binds specific mRNAs such as adenylate cyclase VI (AC6) mRNA for translational repression. Conducting a structure-function analysis, we demonstrated that Bicc1 self-polymerizes through its sterile alpha motif (SAM domain) to form cytoplasmic clusters, which specifically localize and silence bound mRNAs. Altered glucose and lipid metabolism also sustain cystic growth in polycystic kidneys, but the cause of these perturbations is unclear. Here, we found that Bicc1 is essential for normal expression of the gluconeogenic enzymes FBP1 and PEPCK specifically in kidneys, and to maintain euglycemia. In a proteomic interactome screen using a knock-in cell line, we found that TAP-tagged Bicc1 binds the C-Terminal to Lis-Homology domain (CTLH) complex, the mammalian orthologue of the glucose-induced degradation (Gid) complex that triggers degradation of FBP1 in S. cerevisiae. Bicc1 stimulates FBP1 expression in vitro in cultured murine inner medullary collecting duct (mIMCD3) cells, albeit independently of its interaction with the CTLH complex. Instead, Bicc1 and CTLH complex seem to mutually downregulate each other. Bicc1 interactome is also enriched with metabolic related proteins. Here we found that Bicc1 coimmunoprecipitates with rate-limiting metabolic enzymes and contributes to maintain their protein levels, and that deletion of Bicc1 significantly alters the renal metabolome while enhances autophagy. Altogether, these results implicate Bicc1 as an important metabolic regulator in the etiology of polycystic kidneys.

EPFL2017

Analysis of mice with conditional ferritin H deletion

Liviu Vanoaica

Iron is a transitional metal required by virtually all organisms as a dietary micromineral, indispensable for cellular survival and proliferation. The management of iron absorption and distribution in the organism and inside the cell must be tightly regulated in order to avoid the deleterious consequences of free iron-induced oxidative stress. Ferritin is the major protein responsible for iron storage and release of intracellular iron. Ferritin shells are formed from two types of subunits, known as H and L. The present work investigates the effects of conditional ferritin H deletion in mice. A broad ferritin H deletion in liver, spleen, bone marrow and thymus results in an alteration of iron metabolism, characterized by increased transferrin saturation and hepcidin mRNA levels and decreased liver iron deposits. Iron loading prior to deletion leads to liver failure early after deletion, showing that ferritin H is indispensable for limiting iron toxicity through iron sequestration. A hepatocyte-specific deletion fails to reproduce the same phenotype, suggesting an important role for Kupffer macrophages in liver iron detoxification. Ferritin H is also required for B lymphocyte survival, as indicated by the loss of mature B cells in an CD19-specific ferritin H deletion mouse strain, where this population is substantially reduced because of massive reactive oxygen species generation. Conditional deletion in heart leads to fibrosis, increased oxidative stress and to a switch towards a gene expression profile characteristic for cardiac hypertrophy, while iron loading prior to deletion causes a dramatic alteration of the cardiac output function and ultimately to heart failure. An intestine-specific ferritin H deletion results in a typical hemochromatotic phenotype, characterized by increased transferrin saturation and liver iron stores, increased hepcidin expression and decreased IRP2 activity. Hepcidin-mediated ferroportin downregulation at protein level is also shown not to be sufficient to limit the intestinal iron export. An modified version of the current model of intestinal iron absorption is proposed, that includes the function of ferritin H as an iron sink in intestine. This work contributes to a better understanding of iron metabolism in general and intestinal iron absorption in particular, and it might have an impact on the development of treatments against human hemochomatosis and anemia.

EPFL2009