The SIRT1 deacetylase inhibits fat synthesis and stimulates fat oxidation in response to fasting, but the underlying mechanisms remain unclear. Here we report that SREBP-1c, a key lipogenic activator, is an in vivo target of SIRT1. SIRT1 interaction with SREBP-1c was increased during fasting and decreased upon feeding, and consistently, SREBP-1c acetylation levels were decreased during fasting in mouse liver. Acetylated SREBP-1c levels were also increased in HepG2 cells treated with insulin and glucose to mimic feeding conditions, and down-regulation of p300 by siRNA decreased the acetylation. Depletion of hepatic SIRT1 by adenoviral siRNA increased acetylation of SREBP-1c with increased lipogenic gene expression. Tandem mass spectrometry and mutagenesis studies revealed that SREBP-1c is acetylated by p300 at Lys-289 and Lys-309. Mechanistic studies using acetylation-defective mutants showed that SIRT1 deacetylates and inhibits SREBP-1c transactivation by decreasing its stability and its occupancy at the lipogenic genes. Remarkably, SREBP-1c acetylation levels were elevated in dietinduced obese mice, and hepatic overexpression of SIRT1 or treatment with resveratrol, a SIRT1 activator, daily for 1 week decreased acetylated SREBP-1c levels with beneficial functional outcomes. These results demonstrate an intriguing connection between elevated SREBP-1c acetylation and increased lipogenic gene expression, suggesting that abnormally elevated SREBP-1c acetylation increases SREBP-1c lipogenic activity in obese mice. Reducing acetylation of SREBP-1c by targeting SIRT1 may be useful for treating metabolic disorders, including fatty liver, obesity, and type II diabetes.The NAD ϩ -dependent SIRT1 (sirtuin 1) deacetylase plays a critical role in cellular metabolism, stress responses, and possibly aging by modulating the activity of transcription factors and cofactors by protein deacetylation (1-4). In response to low nutritional availability, SIRT1 functions as a master switch to maintain lipid and glucose homeostasis and energy balance by regulating important metabolic regulators, such as PGC-1␣ (PPAR␥ coactivator ␣), Foxo-1, and liver X receptor (1, 5-7). We recently identified the nuclear bile acid receptor, farnesoid X receptor (FXR), 3 as an important in vivo target of SIRT1 in the regulation of hepatic lipid metabolism (8). Of these reported regulators, the function of SIRT1 in deacetylating and enhancing the activity of PGC-1␣ has been well established (1,5,9,10).

Summary The nuclear bile acid receptor FXR is critical for regulation of lipid and glucose metabolism. Here we report that FXR is a target of SIRT1, a deacetylase that mediates nutritional and hormonal modulation of hepatic metabolism. Lysine 217 of FXR is the major acetylation site targeted by p300 and SIRT1. Acetylation of FXR increases its stability but inhibits heterodimerization with RXRα, DNA binding, and transactivation activity. Down-regulation of hepatic SIRT1 increased FXR acetylation with deleterious metabolic outcomes. Surprisingly, in mouse models of metabolic disease, FXR interaction with SIRT1 and p300 was dramatically altered, FXR acetylation levels were elevated, and overexpression of SIRT1 or resveratrol treatment reduced acetylated FXR levels. Our data demonstrate that FXR acetylation is normally dynamically regulated by p300 and SIRT1 but is constitutively elevated in metabolic disease states. Small molecules that inhibit FXR acetylation by targeting SIRT1 or p300 may be promising therapeutic agents for metabolic disorders.

Despite the well-documented association between insulin resistance and cardiovascular disease, the key targets of insulin relevant to the development of cardiovascular disease are not known. Here, using non-biased profiling methods, we identify the enzyme flavin monooxygenase 3 (Fmo3), to be a target of insulin. FMO3 produces trimethylamine N-oxide (TMAO), which has recently been suggested to promote atherosclerosis in mice and humans. We show that FMO3 is suppressed by insulin in vitro, increased in obese/insulin resistant male mice, and increased in obese/insulin resistant humans. Knockdown of FMO3 in insulin resistant mice suppresses FoxO1, a central node for metabolic control, and entirely prevents the development of hyperglycemia, hyperlipidemia and atherosclerosis. Taken together, these data indicate that FMO3 is required for FoxO1 expression and the development of metabolic dysfunction.

SummaryClostridium difficile toxin B (TcdB) is a critical virulence factor causing diseases associated with C. difficile infection (CDI). Here we carried out CRISPR/Cas9-mediated genome-wide screens and identified the members of the Wnt receptor Frizzled (FZDs) family as TcdB receptors. TcdB binds to the conserved Wnt-binding site known as the cysteine-rich domain (CRD), with the highest affinity toward FZD1, 2, and 7. TcdB competes with Wnt for binding to FZDs, and its binding blocks Wnt signaling. FZD1/2/7 triple-knockout (KO) cells are highly resistant to TcdB, and recombinant FZD2-CRD prevented TcdB binding to the colonic epithelium. Colonic organoids cultured from FZD7 KO mice, combined with knock-down of FZD1 and 2, showed increased resistance to TcdB. The colonic epithelium in FZD7 KO mice was less susceptible to TcdB-induced tissue damage in vivo. These findings establish FZDs as physiologically relevant receptors for TcdB in the colonic epithelium.

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Sirtuin 1 (SIRT1) is a NAD-dependent deacetylase that is critically involved in diverse cellular processes including metabolic disease, cancer, and possibly aging. Despite extensive studies on SIRT1 function, how SIRT1 levels are regulated remains relatively unknown. Here, we report that the nuclear bile acid receptor farnesoid X receptor (FXR) inhibits microRNA-34a (miR34a) in the liver, which results in a positive regulation of SIRT1 levels. Activation of FXR by the synthetic agonist GW4064 decreases hepatic miR-34a levels in normal mice, and consistently, hepatic miR-34a levels are elevated in FXR-null mice. FXR induces expression of small heterodimer partner (SHP), an orphan nuclear receptor and transcriptional corepressor, which in turn results in repression of p53, a key activator of the miR34a gene, by inhibiting p53 occupancy at the promoter. MiR-34a decreased SIRT1 levels by binding to the 3-untranslated region of SIRT1 mRNA, and adenovirus-mediated overexpression of miR-34a substantially decreased SIRT1 protein levels in mouse liver. Remarkably, miR-34a levels were elevated, and SIRT1 protein levels were reduced in diet-induced obese mice, and FXR activation in these mice reversed the miR-34a and SIRT1 levels, indicating an intriguing link among FXR activation, decreased miR-34a, and subsequently, increased SIRT1 levels. Our study demonstrates an unexpected role of the FXR/SHP pathway in controlling SIRT1 levels via miR-34a inhibition and that elevated miR-34a levels in obese mice contribute to decreased SIRT1 levels. Manipulation of this regulatory network may be useful for treating diseases of aging, such as metabolic disease and cancer.The NAD ϩ -dependent Sirtuin 1 (SIRT1) 3 deacetylase plays a critical role in cellular metabolism, the stress response, and possibly aging, by modulating the activity of its target proteins via protein deacetylation (1-4). Recent studies demonstrate that SIRT1 plays an important role in maintaining metabolic homeostasis in response to hormonal and nutritional fluctuations by modulating the activity of PGC-1␣, a master metabolic regulator (5, 6). During nutritional deprivation, SIRT1 promotes fat mobilization and suppresses adipogenesis and regulates hepatic glucose and lipid metabolism by activating key metabolic regulators, including PGC-1␣ (5, 6). Activation of SIRT1 by natural or synthetic SIRT1 activators reduced acetylation levels of PGC-1␣ and protected against diet-induced obesity and insulin resistance by promoting mitochondrial function (7-9). SIRT1 levels are dynamically regulated in response to fasting and feeding under physiological conditions but markedly reduced in diet-induced obese mice (10). However, the molecular basis by which SIRT1 levels are regulated under normal conditions and why they are substantially reduced in metabolic disease states remains largely unknown.The nuclear receptor, farnesoid X receptor (FXR), is the primary biosensor for endogenous bile acids and regulates expression of numerous genes involved in lipid and glucose metabolism (...

Dissecting the role of insulin in the complex regulation of triglyceride metabolism is necessary for understanding dyslipidemia and steatosis. Liver Insulin Receptor Knockout (LIRKO) mice show that in the physiological context of feeding, hepatic insulin signaling is not required for the induction of mTORC1, an upstream activator of the lipogenic regulator, SREBP-1c. Feeding induces SREBP-1c mRNA in LIRKO livers, though not to the extent observed in controls. A high fructose diet also partially induces SREBP-1c and lipogenic gene expression in LIRKO livers. Insulin signaling becomes more important in the pathological context of obesity, as knockdown of the insulin receptor in ob/ob mice, a model of Type 2 diabetes, using anti-sense oligonucleotides, abolishes the induction of SREBP-1c and its targets by obesity and ameliorates steatosis. Thus, insulin-independent signaling pathways can partially compensate for insulin in the induction of SREBP-1c by feeding but the further induction by obesity/Type 2 diabetes is entirely dependent upon insulin.

Background: Pancreatic cancer is one of the most lethal human cancers. N 6-methyladenosine (m 6 A), a common eukaryotic mRNA modification, plays critical roles in both physiological and pathological processes. However, its role in pancreatic cancer remains elusive. Methods: LC/MS was used to profile m 6 A levels in pancreatic cancer and normal tissues. Bioinformatics analysis, real-time PCR, immunohistochemistry, and western blotting were used to identify the role of m 6 A regulators in pancreatic cancer. The biological effects of methyltransferase-like 14 (METTL14), an mRNA methylase, were investigated using in vitro and in vivo models. MeRIP-Seq and RNA-Seq were used to assess the downstream targets of METTL14.