Emma De Fabiani

Coordinated Control of Cholesterol Catabolism to Bile Acids and of Gluconeogenesis via a Novel Mechanism of Transcription Regulation Linked to the Fasted-to-fed Cycle

Bile acid metabolism plays an essential role in cholesterol homeostasis and is critical for the initiation of atherosclerotic disease. However, despite the recent advances, the molecular mechanisms whereby bile acids regulate gene transcription and cholesterol homeostasis in mammals still need further investigations. Here, we show that bile acids suppress transcription of the gene (CYP7A1) encoding cholesterol 7α-hydroxylase, the rate-limiting enzyme in bile acid biosynthesis, also through an unusual mechanism not involving the bile acid nuclear receptor, farnesoid X receptor. By performing cell-based reporter assays, protein/protein interaction, and chromatin immunoprecipitation assays, we demonstrate that bile acids impair the recruitment of peroxisome proliferator-activated receptor-γ coactivator-1α and cAMP response element-binding protein-binding protein by hepatocyte nuclear factor-4α, a master regulator of CYP7A1. We also show for the first time that bile acids inhibit transcription of the gene (PEPCK) encoding phosphoenolpyruvate carboxykinase, the rate-limiting enzyme in gluconeogenesis, through the same farnesoid X receptor-independent mechanism. Chromatin immunoprecipitation assay revealed that bile acid-induced dissociation of coactivators from hepatocyte nuclear factor-4α decreased the recruitment of RNA polymerase II to the core promoter and downstream in the 3′-untranslated regions of these two genes, reflecting the reduction of gene transcription. Finally, we found that Cyp7a1 expression was stimulated in fasted mice in parallel to Pepck, whereas the same genes were repressed by bile acids. Collectively, these results reveal a novel regulatory mechanism that controls gene transcription in response to extracellular stimuli and argue that the transcription regulation by bile acids of genes central to cholesterol and glucose metabolism should be viewed dynamically in the context of the fasted-to-fed cycle.

Inhibition of Class I Histone Deacetylases Unveils a Mitochondrial Signature and Enhances Oxidative Metabolism in Skeletal Muscle and Adipose Tissue

Chromatin modifications are sensitive to environmental and nutritional stimuli. Abnormalities in epigenetic regulation are associated with metabolic disorders such as obesity and diabetes that are often linked with defects in oxidative metabolism. Here, we evaluated the potential of class-specific synthetic inhibitors of histone deacetylases (HDACs), central chromatin-remodeling enzymes, to ameliorate metabolic dysfunction. Cultured myotubes and primary brown adipocytes treated with a class I–specific HDAC inhibitor showed higher expression of Pgc-1α, increased mitochondrial biogenesis, and augmented oxygen consumption. Treatment of obese diabetic mice with a class I– but not a class II–selective HDAC inhibitor enhanced oxidative metabolism in skeletal muscle and adipose tissue and promoted energy expenditure, thus reducing body weight and glucose and insulin levels. These effects can be ascribed to increased Pgc-1α action in skeletal muscle and enhanced PPARγ/PGC-1α signaling in adipose tissue. In vivo ChIP experiments indicated that inhibition of HDAC3 may account for the beneficial effect of the class I–selective HDAC inhibitor. These results suggest that class I HDAC inhibitors may provide a pharmacologic approach to treating type 2 diabetes.

Olive Oil Phenols Modulate the Expression of Metalloproteinase 9 in THP-1 Cells by Acting on Nuclear Factor-κB Signaling

In vivo studies suggest that the phenolic component contributes to the anti-inflammatory and antiatherosclerotic actions of olive oil; however, the effects in circulating cells are not fully characterized. Monocytes play a key role in inflammation-based diseases by expressing several molecules, including metalloproteinases (MMPs). In the present study, we investigated the effects of olive oil phenolic extract and individual compounds on MMP-9 in THP-1 cells, a human monocyte-like cell line. Olive oil extract prevented the stimulation of MMP-9 expression and secretion in tumor necrosis factor alpha-treated THP-1 cells. Oleuropein aglycone, a typical olive oil phenol, was active at concentrations found in the extract, although other compounds probably contribute to the biological activity. We also found that the effect of the extract and individual compounds on MMP-9 is due to impaired nuclear factor-kappaB signaling. Our findings provide further evidence on the mechanisms by which olive oil reduces the inflammatory burden associated with disorders, such as atherosclerosis.

Insights in the regulation of cholesterol 7α‐hydroxylase gene reveal a target for modulating bile acid synthesis

The transcription of the gene (CYP7A1) encoding cholesterol 7α‐hydroxylase, a key enzyme in cholesterol homeostasis, is repressed by bile acids via multiple mechanisms involving members of the nuclear receptor superfamily. Here, we describe a regulatory mechanism that can be exploited for modulating bile acid synthesis. By dissecting the mechanisms of CYP7A1 transcription, we found that bile acids stimulate the sequential recruitment of the histone deacetylases (HDACs) 7, 3, and 1, and of the corepressor SMRTα (silencing mediator of retinoid and thyroid receptors‐α) and the nuclear corepressor. Bile acids, but not the farnesoid X receptor–selective agonist GW4064, increase the nuclear concentration of HDAC7, which promotes the assembly of a repressive complex that ultimately represses CYP7A1 transcription. Interestingly, despite its high basal expression level, small heterodimer partner (SHP) is associated with the CYP7A1 promoter only at a later stage of bile acid repression. Gene silencing with small interfering RNA confirms that HDAC7 is the key factor required for the repression of CYP7A1 transcription, whereas knockdown of SHP does not prevent the down‐regulation of CYP7A1. Administration of the HDAC inhibitors valproic acid or trichostatin A to genetically hypercholesterolemic mice increases Cyp7a1 messenger RNA and bile acid synthesis and consequently markedly reduces total plasma and low‐density lipoprotein cholesterol. Conclusion: By using a combination of molecular, cellular, and animal models, our study highlights the importance of HDACs in the feedback regulation of CYP7A1 transcription and identifies these enzymes as potential targets to modulate bile acid synthesis and for the treatment of hypercholesterolemia. (HEPATOLOGY 2007.)

Diabetes-induced myelin abnormalities are associated with an altered lipid pattern: protective effects of LXR activation

Diabetic peripheral neuropathy (DPN) is characterized by myelin abnormalities; however, the molecular mechanisms underlying such deficits remain obscure. To uncover the effects of diabetes on myelin alterations, we have analyzed myelin composition. In a streptozotocin-treated rat model of diabetic neuropathy, analysis of sciatic nerve myelin lipids revealed that diabetes alters myelins phospholipid, FA, and cholesterol content in a pattern that can modify membrane fluidity. Reduced expression of relevant genes in the FA biosynthetic pathway and decreased levels of the transcriptionally active form of the lipogenic factor sterol-regulatory element binding factor-1c (SREBF-1c) were found in diabetic sciatic nerve. Expression of myelins major protein, myelin protein zero (P0), was also suppressed by diabetes. In addition, we confirmed that diabetes induces sciatic nerve myelin abnormalities, primarily infoldings that have previously been associated with altered membrane fluidity. In a diabetic setting, synthetic activator of the nuclear receptor liver X receptor (LXR) increased SREBF-1c function and restored myelin lipid species and P0 expression levels to normal. These LXR-modulated improvements were associated with restored myelin structure in sciatic nerve and enhanced performance in functional tests such as thermal nociceptive threshold and nerve conduction velocity. These findings demonstrate an important role for the LXR-SREBF-1c axis in protection from diabetes-induced myelin abnormalities.

Disruption of the gene encoding 3β‐hydroxysterol Δ14‐reductase (Tm7sf2) in mice does not impair cholesterol biosynthesis

Tm7sf2 gene encodes 3β‐hydroxysterol Δ14‐reductase (C14SR, DHCR14), an endoplasmic reticulum enzyme acting on Δ14‐unsaturated sterol intermediates during the conversion of lanosterol to cholesterol. The C‐terminal domain of lamin B receptor, a protein of the inner nuclear membrane mainly involved in heterochromatin organization, also possesses sterol Δ14‐reductase activity. The subcellular localization suggests a primary role of C14SR in cholesterol biosynthesis. To investigate the role of C14SR and lamin B receptor as 3β‐hydroxysterol Δ14‐reductases, Tm7sf2 knockout mice were generated and their biochemical characterization was performed. No Tm7sf2 mRNA was detected in the liver of knockout mice. Neither C14SR protein nor 3β‐hydroxysterol Δ14‐reductase activity were detectable in liver microsomes of Tm7sf2(−/−) mice, confirming the effectiveness of gene inactivation. C14SR protein and its enzymatic activity were about half of control levels in the liver of heterozygous mice. Normal cholesterol levels in liver membranes and in plasma indicated that, despite the lack of C14SR, Tm7sf2(−/−) mice are able to perform cholesterol biosynthesis. Lamin B receptor 3β‐hydroxysterol Δ14‐reductase activity determined in liver nuclei showed comparable values in wild‐type and knockout mice. These results suggest that lamin B receptor, although residing in nuclear membranes, may contribute to cholesterol biosynthesis in Tm7sf2(−/−) mice. Affymetrix microarray analysis of gene expression revealed that several genes involved in cell‐cycle progression are downregulated in the liver of Tm7sf2(−/−) mice, whereas genes involved in xenobiotic metabolism are upregulated.

Enterodiol and Enterolactone Modulate the Immune Response by Acting on Nuclear Factor-κB (NF-κB) Signaling

Lignan-rich whole-grain cereals, beans, berries, and nuts show protective effects against a variety of chronic diseases, including cancer. Lignans are converted by intestinal microflora to enterolactone (EL) and its oxidation product enterodiol (ED). To investigate the immunomodulatory effect of EL and ED in human cells, peripheral blood lymphocytes were treated with increasing physiologically relevant concentrations of EL and ED (0-1000 microM) and stimulated with lipopolysaccharide (LPS) and anti-CD3 plus anti-CD28 monoclonal antibodies. A dose-related inhibition of cell proliferation and cytokine production was observed, with EL being the most active. Molecular investigations in THP-1 cells showed that both EL and ED prevented inhibitory-kappaB (I-kappaB) degradation and nuclear factor-kappaB (NF-kappaB) activation, which in turn resulted in decreased tumor necrosis factor-alpha (TNF-alpha) production. EL and ED were also able to pass the intestinal barrier and modulate cytokine production. The findings of the present study reveal potential mechanisms that could explain some in vivo beneficial effects of lignans.

Linking epigenetics to lipid metabolism: Focus on histone deacetylases

Abstract A number of recent studies revealed that epigenetic modifications play a central role in the regulation of lipid and of other metabolic pathways such as cholesterol homeostasis, bile acid synthesis, glucose and energy metabolism. Epigenetics refers to aspects of genome functions regulated in a DNA sequence-independent fashion. Chromatin structure is controlled by epigenetic mechanisms through DNA methylation and histone modifications. The main modifications are histone acetylation and deacetylation on specific lysine residues operated by two different classes of enzymes: Histone acetyltransferases (HATs) and histone deacetylases (HDACs), respectively. The interaction between these enzymes and histones can activate or repress gene transcription: Histone acetylation opens and activates chromatin, while deacetylation of histones and DNA methylation compact chromatin making it transcriptionally silent. The new evidences on the importance of HDACs in the regulation of lipid and other metabolic pathways will open new perspectives in the comprehension of the pathophysiology of metabolic disorders.

The lipogenic regulator Sterol Regulatory Element Binding Factor-1c is required to maintain peripheral nerve structure and function.

Myelin is a membrane characterized by high lipid content to facilitate impulse propagation. Changes in myelin fatty acid (FA) composition have been associated with peripheral neuropathy [1], but the specific role of peripheral nerve FA synthesis in myelin formation and function is poorly understood. We explored the extent to which lack of the key regulator of FA synthesis as Sterol Regulatory Element Binding Factor-1c (Srebf-1c) could result in the development of peripheral neuropathy. We found that Srebf-1c null mice display a neuropathic phenotype consisting in hypermyelinated small caliber fibers, the result of changes in myelin periodicity. Unexpectedly, transcriptomics and metabolomics revealed activation of peroxisome proliferator activated receptor α (Pparα) signaling in Srebf-1c null peripheral nerve as a result of increased levels of two distinct phosphatidylcholine-based Pparα ligands, PC-C16:0/C18:1 and PC-C18:0/C18:1 [2, 3]. Pparα is a nuclear receptor that directs uptake, utilization and catabolism of FAs [4]. As a consequence of abnormal local Pparα activation, Srebf-1c null peripheral nerve exhibit increased fatty acid utilization, a detrimental condition leading to peripheral neuropathy. Treatment with a Pparα antagonist rescues the neuropathy of Srebf-1c null mice. These findings reveal the importance of FA synthesis to sustain peripheral nerve structure and function.

The sirtuin class of histone deacetylases: Regulation and roles in lipid metabolism

After the completion of the human genome sequence and that from many other organisms, last decade has witnessed a spectacular gain of knowledge on gene functions. These studies provided new insights on the roles of genes in physiology and disease. Nonetheless, the availability of genetically modified models and of “omics” technologies such as next generation sequencing unveiled clear evidences on epigenetic regulation of many cellular functions. At this regard, sirtuins, belonging to class III histone deacetylase family, have emerged as regulators of metabolism as well as other cellular processes and seem ideally suited as targets of future therapeutical interventions. This review deals on general aspects of the biology of sirtuins and focuses on their relevance in lipid metabolism in different tissues, pointing to their exploitation as potential pharmacological targets of compounds that could be used as new therapeutic alternatives in several disorders ranging from type 2 diabetes and obesity to age‐related cardiovascular and neurodegenerative diseases.