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Exposure to cigarette smoke (CS) causes detrimental health effects, increasing the risk of cardiovascular, pulmonary diseases and carcinogenesis in exposed individuals. The impact of CS on Inflammatory Bowel Disease (IBD) has been established by a number of epidemiological and clinical studies. In fact, CS is associated with a higher risk of developing Crohn’s disease (CD) while inversely correlates with the development, disease risks, and relapse rate of ulcerative colitis (UC). To investigate the effect of CS exposure on experimental colitis, we performed a comprehensive and integrated comparative analysis of colon transcriptome and microbiome in mice exposed to dextran sodium sulfate (DSS) and CS. Colon transcriptome analysis revealed that CS downregulated specific pathways in a concentration-dependent manner, affecting both the inflammatory state and composition of the gut microbiome. Metagenomics analysis demonstrated that CS can modulate DSS-induced dysbiosis of specific bacterial genera, contributing to resolve the inflammation or accelerate recovery. The risks of smoking far outweigh any possible benefit, thus smoking cessation must always be encouraged because of its significant health benefits. However, the inverse association between active smoking and the development of UC cannot be ignored and the present study lays the foundation for investigating potential molecular mechanisms responsible for the attenuation of colitis by certain compounds of tobacco when decoupled from combustion.

Inflammatory bowel disease (IBD) represents a group of progressive disorders characterized by recurrent chronic inflammation of the gut. Ulcerative colitis and Crohn′s disease are the major manifestations of IBD. While our understanding of IBD has progressed in recent years, its etiology is far from being fully understood, resulting in suboptimal treatment options. Complementing other biological endpoints, bioanalytical “omics” methods that quantify many biomolecules simultaneously have great potential in the dissection of the complex pathogenesis of IBD. In this review, we focus on the rapidly evolving proteomics and lipidomics technologies and their broad applicability to IBD studies; these range from investigations of immune-regulatory mechanisms and biomarker discovery to studies dissecting host–microbiome interactions and the role of intestinal epithelial cells. Future studies can leverage recent advances, including improved analytical methodologies, additional relevant sample types, and integrative multi-omics analyses. Proteomics and lipidomics could effectively accelerate the development of novel targeted treatments and the discovery of complementary biomarkers, enabling continuous monitoring of the treatment response of individual patients; this may allow further refinement of treatment and, ultimately, facilitate a personalized medicine approach to IBD.

The liver is one of the most important organs involved in elimination of xenobiotic and potentially toxic substances. Cigarette smoke (CS) contains more than 7000 chemicals, including those that exert biological effects and cause smoking-related diseases. Though CS is not directly hepatotoxic, a growing body of evidence suggests that it may exacerbate pre-existing chronic liver disease. In this study, we integrated toxicological endpoints with molecular measurements and computational analyses to investigate effects of exposures on the livers of Apoe−/− mice. Mice were exposed to 3R4F reference CS, to an aerosol from the Tobacco Heating System (THS) 2.2, a candidate modified risk tobacco product (MRTP) or to filtered air (Sham) for up to 8 months. THS2.2 takes advantage of a “heat-not-burn” technology that, by heating tobacco, avoids pyrogenesis and pyrosynthesis. After CS exposure for 2 months, some groups were either switched to the MRTP or filtered air. While no group showed clear signs of hepatotoxicity, integrative analysis of proteomics and transcriptomics data showed a CS-dependent impairment of specific biological networks. These networks included lipid and xenobiotic metabolism and iron homeostasis that likely contributed synergistically to exacerbating oxidative stress. In contrast, most proteomic and transcriptomic changes were lower in mice exposed to THS2.2 and in the cessation and switching groups compared to the CS group. Our findings elucidate the complex biological responses of the liver to CS exposure. Furthermore, they provide evidence that THS2.2 aerosol has reduced biological effects, as compared with CS, on the livers of Apoe−/− mice.

We have recently shown that the transcriptional coactivator PGC1α, a master regulator of mitochondrial biogenesis and function, is involved in the control of the intestinal epithelium cell fate. Furthermore, PGC1α protects against colon cancer formation by promoting ROS accumulation and, consequently, mitochondria-mediated apoptosis. Here we provide an additional mechanistic insight into the tumor suppressor activity of PGC1α showing that its pro-apoptotic effect is mediated by Bax. In fact, PGC1α overexpression in HCT116 Bax (-/-) colorectal cancer cells stimulates mitochondrial production and activity, but it fails to induce cell death as well as to oppose tumor growth in the xenograft model. The lack of ROS accumulation in the Bax (-/-) cells strengthens our view that the PGC1α-induced oxidative burst represents one of the main apoptosis-driving factors in colorectal cancer cells.

Peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC1α) is a transcriptional coactivator able to up-regulate mitochondrial biogenesis, respiratory capacity, oxidative phosphorylation, and fatty acid β-oxidation with the final aim of providing a more efficient pathway for aerobic energy production. In the continuously renewed intestinal epithelium, proliferative cells in the crypts migrate along the villus axis and differentiate into mature enterocytes, increasing their respiratory capacity and finally undergoing apoptosis. Here we show that in the intestinal epithelial surface, PGC1α drives mitochondrial biogenesis and respiration in the presence of reduced antioxidant enzyme activities, thus determining the accumulation of reactive oxygen species and fostering the fate of enterocytes toward apoptosis. Combining gain- and loss-of-function genetic approaches in human cells and mouse models of intestinal cancer, we present an intriguing scenario whereby PGC1α regulates enterocyte cell fate and protects against tumorigenesis.

Summary Several steps of the HDL-mediated reverse cholesterol transport (RCT) are transcriptionally regulated by the nuclear receptors LXRs in the macrophages, liver, and intestine. Systemic LXR activation via synthetic ligands induces RCT but also causes increased hepatic fatty acid synthesis and steatosis, limiting the potential therapeutic use of LXR agonists. During the last few years, the participation of the intestine in the control of RCT has appeared more evident. Here we show that while hepatic-specific LXR activation does not contribute to RCT, intestinal-specific LXR activation leads to decreased intestinal cholesterol absorption, improved lipoprotein profile, and increased RCT in vivo in the absence of hepatic steatosis. These events protect against atherosclerosis in the background of the LDLR-deficient mice. Our study fully characterizes the molecular and metabolic scenario that elects the intestine as a key player in the LXR-driven protective environment against cardiovascular disease.

Cholesterol homeostasis is critical for cellular proliferation. Liver X receptor (LXR) alpha and beta are the nuclear receptors responsible for regulation of cholesterol metabolism. In physiological conditions, high intracellular cholesterol levels cause increased synthesis of oxysterols, which activate LXR, thus triggering a transcriptional response for cholesterol secretion and catabolism. Here we employed a mouse model of partial hepatectomy (PH) to dissect the molecular pathways connecting cholesterol homeostasis, cellular proliferation, and LXR. First, we show that hepatic cholesterol content increases after PH, whereas the entire LXR transcriptome is down-regulated. Although LXR messenger RNA (mRNA) levels are unmodified, LXR target genes are significantly down-regulated on day 1 after PH and restored to control levels on day 7, when the liver reaches normal size. The inactivation of LXR following PH is related to the reduced oxysterol availability by way of decreased synthesis, and increased sulfation and secretion. On the contrary, cholesterol synthesis is up-regulated, and extracellular matrix remodeling is enhanced. Second, we show that reactivation of LXR by way of a synthetic ligand determines a negative modulation of hepatocyte proliferation. This effect is sustained by the reactivation of hepatic cholesterol catabolic and secretory pathways, coupled with a significant reduction of cholesterol biosynthesis. Our data unveil a previously unrecognized and apparently paradoxical scenario of LXR modulation. During liver regeneration LXR activity is abated in spite of increasing intracellular cholesterol levels. Turning off LXR-transcriptional pathways is crucial to guaranteeing the requisite intracellular cholesterol levels of regenerating hepatocytes. In line with this hypothesis, pharmacological LXR reactivation during PH significantly reduces liver regeneration capacity.

Bile formation springs from an extraordinary sophisticated secretory network, which combines the activity of transport proteins with the physico-chemical properties of small albeit powerful lipophilic compounds. This robust interplay is activated in response to dietary stimuli, circadian rhythms, and metabolic demands to regulate cholesterol disposal, lipid digestion and absorption in the enterohepatic system. As a result, bile flow is a complex multi-organ effort that requires an integrated flux of information between liver and intestine. A coordinate regulatory task is achieved by nuclear receptors, which are ligand activated transcription factors, responsible for the coherent activation of sets of genes involved in multiple physiological actions, including hepato-biliary homeostasis. Mastering the molecular pathways underlying functional and pharmacological modulation of bile flow has great translational value for potential future treatment of cholestasis and cholelithiasis. In this review, we focus on recent discoveries in the functional biology of bile formation with the explicit aim of underlining their putative clinical relevance.

The metabolic syndrome is an emerging global epidemic characterized by clustering of metabolic abnormalities leading to increased cardiovascular risk: glucose intolerance or type 2 diabetes, dyslipidemia, hypertension, and "central" obesity. Scientists are decoding and piecing together the molecular texture underlying the metabolic syndrome: insulin resistance and dyslipidemia stand out as central pathophysiological events. In this picture, the liver rises as the leading organ in the maintenance of metabolic fitness; it serves as the first relay station for processing dietary information, and encloses the whole biochemical machinery for both glucose and lipid storage and disposal. In addition, the liver is a target of the different endocrine molecules secreted by pancreatic beta-cells and adipose tissue. Evidence collected in animal models supports the central role of the liver in the metabolic syndrome. While specific bereft of insulin sensitivity in skeletal muscle and adipose tissue fails to induce diabetes at certain extent, this is constantly the outcome in case of hepatic insulin resistance. Also, dyslipidemia is currently interpreted as the result of increased flux of free fatty acids to the liver with ensuing misbalance of lipoprotein synthesis and removal. In this review we bring together recent advances in the field of lipid sensing nuclear receptors, adipokines and other molecules responsible for metabolic fitness, and provide a putative coherent frame to conceive the pathophysiology of the metabolic syndrome.

Background & Aims: Studies of the transcriptional networks that regulate nuclear receptor-mediated proliferation of quiescent hepatocytes could lead to new information about liver growth and hepatoprotective strategies. Methods: We used quantitative real-time PCR to analyze expression of neuron-derived orphan receptor 1 (Nor-1) and its target genes during liver regeneration after hepatectomy in mice, and in hepatocellular carcinoma (HCC) samples from patients. We used adenoviral vectors to express Nor-1 in normal liver (Ad/CMV/V5-Nor-1), or reduce its level with small hairpin RNAs (Ad/BLOCK-iT/Nor-1small hairpin RNA) after partial hepatectomy. Results: Levels of Nor-1 messenger RNA and protein, and transcription of Nor-1 target genes (Ccnd1 and Vcam-1), increased during the late priming and proliferative phases of liver regeneration after partial hepatectomy. Levels of NOR-1 messenger RNA and transcription of its target gene CCND1 and of the NOR-1 subfamily member NUR-77 also increased in human HCC samples compared with paired HCC-free tissue. Ad-Nor-1small hairpin RNA reduced the hepatocyte proliferation after hepatectomy. Overexpression of Nor-1 in normal livers of mice induced proliferation of quiescent hepatocytes independently of interleukin-6 and tumor necrosis factor-α signaling. In gene expression profile analysis, Nor-1 altered expression of genes involved in the cell cycle, proliferation, and tumorigenesis. Conclusions: In mice, the orphan nuclear receptor Nor-1 activates proliferation of quiescent hepatocytes and is required for hepatocyte proliferation after partial hepatectomy. Nor-1 and its gene targets are also up-regulated in human HCC samples. Nor-1 activates a transcriptional program that induces hepatocyte proliferation independently of inflammatory signaling pathways. © 2013 AGA Institute.

Objective— Liver X receptors (LXRα, LXRβ) are master regulators of cholesterol homeostasis. In the endothelium, perturbations of cell cholesterol have an impact on fundamental processes. We, therefore, assessed the effects of LXR activation on endothelial functions related to angiogenesis in vitro and in vivo. Methods and Results— LXR agonists (T0901317, GW3965) blunted migration, tubulogenesis, and proliferation of human umbilical vein endothelial cells. By affecting endothelial cholesterol homeostasis, LXR activation impaired the compartmentation of vascular endothelial growth factor receptor-2 in lipid rafts/caveolae and led to defective phosphorylation and downstream signaling of vascular endothelial growth factor receptor-2 upon vascular endothelial growth factor-A stimulation. Consistently, the antiangiogenic actions of LXR agonists could be prevented by coadministration of exogenous cholesterol. LXR agonists reduced endothelial sprouting from wild-type but not from LXRα −/− /LXRβ −/− knockout aortas and blunted the vascularization of implanted angioreactors in vivo. Furthermore, T0901317 reduced the growth of Lewis lung carcinoma grafts in mice by impairing angiogenesis. Conclusion— Pharmacological activation of endothelial LXRs reduces angiogenesis by restraining cholesterol-dependent vascular endothelial growth factor receptor-2 compartmentation and signaling. Thus, administration of LXR agonists could exert therapeutic effects in pathological conditions characterized by uncontrolled angiogenesis.

The G protein-coupled receptor TGR5 has been identified as an important component of the bile acid signaling network, and its activation has been linked to enhanced energy expenditure and improved glycemic control. Here, we demonstrate that activation of TGR5 in macrophages by 6 alpha-ethyl-23(S)-methylcholic acid (6-EMCA, INT-777), a semisynthetic BA, inhibits proinflammatory cytokine production, an effect mediated by TGR5-induced cAMP signaling and subsequent NF-kappa B inhibition. TGR5 activation attenuated atherosclerosis in Ld/r(-/-)Tgr5(+/+) mice but not in Ld/r(-/-)Tgr5(-/-) double-knockout mice. The inhibition of lesion formation was associated with decreased intraplaque inflammation and less plaque macrophage content. Furthermore, Ld/r(-/-)animals transplanted with Tgr5(-/-) bone marrow did not show an inhibition of atherosclerosis by INT-777, further establishing an important role of leukocytes in INT-777-mediated inhibition of vascular lesion formation. Taken together, these data attribute a significant immune modulating function to TGR5 activation in the prevention of atherosclerosis, an important facet of the metabolic syndrome.

Figure 3. PPARβ/δ protein is suppressed in murine regenerating liver (A) and human HCC (C), and negatively correlates with PCNA (B).Anti-PPARβ/δ immunostaining in samples of LR (4–5/group) and 9 paraffin-embedded samples of HCC paired with reference tumor-free tissues (selection: 3 of 9 subjects); PPARβ/δ positive cells (percentage of marked area) were quantified using the ImageJ software. Results are shown as means ± SEM or means (bars) and cases (each line representing paired tumor-free tissue vs . tumor sample). Statistical significance (p≤0.05) assessed by the Kruskal-Wallis One-Way ANOVA on Ranks plus Nemenyi-Damico-Wolfe-Dunn post-hoc test (LR), Wilcoxon Signed-Rank Test (HCC), and Pearson's correlation coefficient (correlation). Legend: lower case letters indicate statistical significance (“a” means reference group; “b” means different from “a”; “c” means different from “a” and “b”); (white) quiescent; (black) proliferating; (▴) Sham; (▪) 0.5 days; (•) 1 day; (♦) 3 days; (▾) 7 dayshttp://dx.doi.org/10.1371/journal.pone.0104449.g003 Mice tissue specimens were fixed in 10% formalin for 12–24 hours, dehydrated and paraffin embedded. We also performed immunohistochemistry on samples of paraffin-embedded HCC ( vs. paired normal tissues; n = 9) received from Creative Bioarray, USA (http://www.creative-bioarray.com/Contact-Us.html). Standard Immunohistochemistry protocols were performed[15]. Briefly, 5 µm-thick sections were treated with 3% hydrogen peroxide for 5 min and with the Dako Cytomation Biotin blocking system (Dako, Denmark) to quench endogenous peroxidase and biotin respectively. Sections were sequentially incubated for 60 min at room temperature in 50% non-immune serum in PBS (to avoid unspecific signals) and overnight at 4°C with the primary antibody (anti-Pcna, Santa Cruz Biotechnology, Santa Cruz, CA; Abcam Anti-PPAR delta antibody, Cat AB23673). Sections were then washed for 10 min in PBS, and incubated for 30 min at room temperature with the secondary biotinylated antibody (Vector Laboratories). After several washing steps with PBS (3 washes 5 min/each), sections were incubated with the avidin-biotin complex (Vector Laboratories) for 30 min at room temperature. After washing in PBS, the peroxidase reaction was initiated by incubation with DAB (Sigma-Aldrich, Milano, Italy). Coverslips were mounted with Permount and evaluated under a light microscope. All the stained sections were analyzed through a confocal microscope (Magnitude: 20×). For each sample, 5 representative images were taken. Number and intensity of marked nuclei were quantified using ImageJ software as previously described [29].

Background & Aims Liver X receptors (LXRs) are transcriptional regulators of cholesterol metabolism, controlling cholesterol flow into cells, catabolism, and efflux. Cholesterol controls cell proliferation; disruptions in cholesterol metabolism have been associated with the development of colon cancer. We investigated whether expression of activated LXR protects against intestinal tumorigenesis in mice. Methods We analyzed the development of colon cancer in mice that express a constitutive active form of LXRα only in the intestinal epithelium, under the control of villin promoter (iVP16LXRα). These mice were crossed with adenomatous polyposis coli (Apc) min/+ mice, or given azoxymethane followed by dextran sodium sulfate, to assess intestinal tumor formation. We also assessed proliferation and apoptosis of a human colorectal cancer cell line (HT29) transfected with an adenoviral vector that expressed Ad VP16hLXRα, compared with cells expressing AdVP16 (control), and their ability to form xenograft tumors in mice. HT29 cells also were incubated with the LXR ligand GW3965. Results In human colorectal cancer cells, ligand-induced activation of LXR or transfection with Ad VP16hLXRα blocked the G1 phase, increased caspase-dependent apoptosis, and slowed growth of xenograft tumors in mice. iVP16LXRα mice formed fewer, smaller tumors than VP16 (control) mice after administration of azoxymethane and dextran sodium sulfate. APC min/+ /iVP16LXRα mice also developed fewer, smaller intestinal tumors than APC min/+ /iVP16 mice. Gene expression analysis indicated that activation of LXRα affected lipid metabolic networks and increased cholesterol efflux in the intestine. Conclusions Expression of activated LXRα blocks proliferation of human colorectal cancer cells and slows the growth of xenograft tumors in mice. It also reduces intestinal tumor formation after administration of chemical carcinogens, and in Apc min/+ mice. LXR agonists therefore might be developed as therapeutic treatments for colorectal cancer.

The nuclear receptor REV-ERBα shapes the daily activity profile of Sterol Response Element Binding Protein (SREBP) and thereby participates in the circadian control of cholesterol and bile acid synthesis in the liver., In mammals, many aspects of behavior and physiology, and in particular cellular metabolism, are coordinated by the circadian timing system. Molecular clocks are thought to rely on negative feedback loops in clock gene expression that engender oscillations in the accumulation of transcriptional regulatory proteins, such as the orphan receptor REV-ERBα. Circadian transcription factors then drive daily rhythms in the expression of clock-controlled output genes, for example genes encoding enzymes and regulators of cellular metabolism. To gain insight into clock output functions of REV-ERBα, we carried out genome-wide transcriptome profiling experiments with liver RNA from wild-type mice, Rev-erbα knock-out mice, or REV-ERBα overexpressing mice. On the basis of these genetic loss- and gain-of-function experiments, we concluded that REV-ERBα participates in the circadian modulation of sterol regulatory element-binding protein (SREBP) activity, and thereby in the daily expression of SREBP target genes involved in cholesterol and lipid metabolism. This control is exerted via the cyclic transcription of Insig2, encoding a trans-membrane protein that sequesters SREBP proteins to the endoplasmic reticulum membranes and thereby interferes with the proteolytic activation of SREBPs in Golgi membranes. REV-ERBα also participates in the cyclic expression of cholesterol-7α-hydroxylase (CYP7A1), the rate-limiting enzyme in converting cholesterol to bile acids. Our findings suggest that this control acts via the stimulation of LXR nuclear receptors by cyclically produced oxysterols. In conclusion, our study suggests that rhythmic cholesterol and bile acid metabolism is not just driven by alternating feeding–fasting cycles, but also by REV-ERBα, a component of the circadian clockwork circuitry., Author Summary The mammalian circadian timing system has a hierarchical architecture: a central pacemaker in the brain's suprachiasmatic nucleus (SCN) synchronizes subsidiary oscillators present in most peripheral cell types. In both SCN neurons and peripheral cells, circadian oscillators are thought to rely on two negative feedback loops. A major feedback loop involves the two cryptochromes CRY1 and CRY2 and the two period proteins PER1 and PER2, which serve as transcriptional repressors for their own genes. An accessory feedback loop couples the expression and activity of the transcriptional activators CLOCK and BMAL1 to the expression of cryptochrome and period proteins. The orphan nuclear receptor REV-ERBα is a key player in this accessory feedback loop, in that it periodically represses Bmal1 transcription. In liver, molecular clocks mediate the temporal gating of metabolic processes. Here we demonstrate that hepatocyte clocks participate in the control of cholesterol and bile acid homeostasis. According to this scenario, REV-ERBα shapes the circadian expression pattern of insulin-induced gene 2 (INSIG2), a resident protein of the endoplasmic reticulum that interferes with the proteolytic activation of sterol response element binding proteins (SREBPs). In turn SREBPs govern the rhythmic expression of enzymes with key functions in sterol and fatty acid synthesis. The circadian production of sterols (in particular oxysterols) may engender the cyclic activation of LXR nuclear receptors, which serve as critical activators of Cyp7a1 transcription. CYP7A1, also known as cholesterol 7α-hydroxylase, catalyzes the rate-limiting step in bile acid synthesis.

The biological effects of urolithins remain poorly characterized, despite wide-spread human exposure via the dietary consumption of their metabolic precursors, the ellagitannins, which are found in the pomegranate fruit, as well as in nuts and berries. We identified urolithin A (UA) as a first-in-class natural compound that induces mitophagy both in vitro and in vivo following oral consumption. In C. elegans, UA prevented the accumulation of dysfunctional mitochondria with age and extended lifespan. Likewise, UA prolonged normal activity during aging in C. elegans, including mobility and pharyngeal pumping, while maintaining mitochondrial respiratory capacity. These effects translated to rodents, where UA improved exercise capacity in two different mouse models of age-related decline of muscle function, as well as in young rats. Our findings highlight the health benefits of urolithin A and its potential application in strategies to improve mitochondrial and muscle function.