Cyclo His-Pro Attenuates Muscle Degeneration in Murine Myopathy Models.

Among the inherited myopathies, a group of muscular disorders characterized by structural and metabolic impairments in skeletal muscle, Duchenne muscular dystrophy (DMD) stands out for its devastating progression. DMD pathogenesis is driven by the progressive degeneration of muscle fibers, resulting in inflammation and fibrosis that ultimately affect the overall muscle biomechanics. At the opposite end of the spectrum of muscle diseases, age-related sarcopenia is a common condition that affects an increasing proportion of the elderly. Although characterized by different pathological mechanisms, DMD and sarcopenia share the development of progressive muscle weakness and tissue inflammation. Here, the therapeutic effects of Cyclo Histidine-Proline (CHP) against DMD and sarcopenia are evaluated. In the mdx mouse model of DMD, it is shown that CHP restored muscle contractility and force production, accompanied by the reduction of fibrosis and inflammation in skeletal muscle. CHP furthermore prevented the development of cardiomyopathy and fibrosis in the diaphragm, the two leading causes of death for DMD patients. CHP also attenuated muscle atrophy and functional deterioration in a mouse model of age-related sarcopenia. These findings from two different models of muscle dysfunction hence warrant further investigation into the effects of CHP on muscle pathologies in animal models and eventually in patients.

Inhibition of chloride intracellular channel protein 1 (CLIC1) ameliorates liver fibrosis phenotype by activating the Ca2+-dependent Nrf2 pathway.

Persistent damage to liver cells leads to liver fibrosis, which is characterized by the accumulation of scar tissue in the liver, ultimately leading to cirrhosis and serious complications. Because it is difficult to reverse cirrhosis once it has progressed, the primary focus has been on preventing the progression of liver fibrosis. However, studies on therapeutic agents for liver fibrosis are still lacking. Here, we investigated that the natural dipeptide cyclic histidine-proline (CHP, also known as diketopiperazine) shows promising potential as a therapeutic agent in models of liver injury by inhibiting the progression of fibrosis through activation of the Nrf2 pathway. To elucidate the underlying biological mechanism of CHP, we used the Cellular Thermal Shift Assay (CETSA)-LC-MS/MS, a label-free compound-based target identification platform. Chloride intracellular channel protein 1 (CLIC1) was identified as a target whose thermal stability is increased by CHP treatment. We analyzed the direct interaction of CHP with CLIC1 which revealed a potential interaction between CHP and the E228 residue of CLIC1. Biological validation experiments showed that knockdown of CLIC1 mimicked the antioxidant effect of CHP. Further investigation using a mouse model of CCl4-induced liver fibrosis in wild-type and CLIC1 KO mice revealed the critical involvement of CLIC1 in mediating the effects of CHP. Taken together, our results provide evidence that CHP exerts its anti-fibrotic effects through specific binding to CLIC1. These insights into the mechanism of action of CHP may pave the way for the development of novel therapeutic strategies for fibrosis-related diseases.

Cyclo(His-Pro): A further step in the management of steatohepatitis.

Background & Aims: Non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH) have become the world's most common liver diseases, placing a growing strain on healthcare systems worldwide. Nonetheless, no effective pharmacological treatment has been approved. The naturally occurring compound cyclo histidine-proline (His-Pro) (CHP) is an interesting candidate for NAFLD management, given its safety profile and anti-inflammatory effects. Methods: Two different mouse models of liver disease were used to evaluate protective effects of CHP on disease progression towards fibrosis: a model of dietary NAFLD/NASH, achieved by thermoneutral housing (TN) in combination with feeding a western diet (WD), and liver fibrosis caused by repeated injections with carbon tetrachloride (CCl4). Results: Treatment with CHP limited overall lipid accumulation, lowered systemic inflammation, and prevented hyperglycaemia. Histopathology and liver transcriptomics highlighted reduced steatosis and demonstrated remarkable protection from the development of inflammation and fibrosis, features which herald the progression of NAFLD. We identified the extracellular signal-regulated kinase (ERK) pathway as an early mediator of the cellular response to CHP. Conclusions: CHP was active in both the preventive and therapeutic setting, reducing liver steatosis, fibrosis, and inflammation and improving several markers of liver disease. Impact and implications: Considering the incidence and the lack of approved treatments, it is urgent to identify new strategies that prevent and manage NAFLD. CHP was effective in attenuating NAFLD progression in two animal models of the disease. Overall, our work points to CHP as a novel and effective strategy for the management of NAFLD, fuelling optimism for potential clinical studies.

Upregulation of the ERRγ-VDAC1 axis underlies the molecular pathogenesis of pancreatitis.

Emerging evidence suggest that transcription factors play multiple roles in the development of pancreatitis, a necroinflammatory condition lacking specific therapy. Estrogen-related receptor γ (ERRγ), a pleiotropic transcription factor, has been reported to play a vital role in pancreatic acinar cell (PAC) homeostasis. However, the role of ERRγ in PAC dysfunction remains hitherto unknown. Here, we demonstrated in both mice models and human cohorts that pancreatitis is associated with an increase in ERRγ gene expression via activation of STAT3. Acinar-specific ERRγ haploinsufficiency or pharmacological inhibition of ERRγ significantly impaired the progression of pancreatitis both in vitro and in vivo. Using systematic transcriptomic analysis, we identified that voltage-dependent anion channel 1 (VDAC1) acts as a molecular mediator of ERRγ. Mechanistically, we showed that induction of ERRγ in cultured acinar cells and mouse pancreata enhanced VDAC1 expression by directly binding to specific site of the Vdac1 gene promoter and resulted in VDAC1 oligomerization. Notably, VDAC1, whose expression and oligomerization were dependent on ERRγ, modulates mitochondrial Ca2+ and ROS levels. Inhibition of the ERRγ-VDAC1 axis could alleviate mitochondrial Ca2+ accumulation, ROS formation and inhibit progression of pancreatitis. Using two different mouse models of pancreatitis, we showed that pharmacological blockade of ERRγ-VDAC1 pathway has therapeutic benefits in mitigating progression of pancreatitis. Likewise, using PRSS1R122H-Tg mice to mimic human hereditary pancreatitis, we demonstrated that ERRγ inhibitor also alleviated pancreatitis. Our findings highlight the importance of ERRγ in pancreatitis progression and suggests its therapeutic intervention for prevention and treatment of pancreatitis.

CycloZ Improves Hyperglycemia and Lipid Metabolism by Modulating Lysine Acetylation in KK-Ay Mice.

BACKGRUOUND: CycloZ, a combination of cyclo-His-Pro and zinc, has anti-diabetic activity. However, its exact mode of action remains to be elucidated. METHODS: KK-Ay mice, a type 2 diabetes mellitus (T2DM) model, were administered CycloZ either as a preventive intervention, or as a therapy. Glycemic control was evaluated using the oral glucose tolerance test (OGTT), and glycosylated hemoglobin (HbA1c) levels. Liver and visceral adipose tissues (VATs) were used for histological evaluation, gene expression analysis, and protein expression analysis. RESULTS: CycloZ administration improved glycemic control in KK-Ay mice in both prophylactic and therapeutic studies. Lysine acetylation of peroxisome proliferator-activated receptor gamma coactivator 1-alpha, liver kinase B1, and nuclear factor-κB p65 was decreased in the liver and VATs in CycloZ-treated mice. In addition, CycloZ treatment improved mitochondrial function, lipid oxidation, and inflammation in the liver and VATs of mice. CycloZ treatment also increased the level of ß-nicotinamide adenine dinucleotide (NAD+), which affected the activity of deacetylases, such as sirtuin 1 (Sirt1). CONCLUSION: Our findings suggest that the beneficial effects of CycloZ on diabetes and obesity occur through increased NAD+ synthesis, which modulates Sirt1 deacetylase activity in the liver and VATs. Given that the mode of action of an NAD+ booster or Sirt1 deacetylase activator is different from that of traditional T2DM drugs, CycloZ would be considered a novel therapeutic option for the treatment of T2DM.

Amelioration of obesity-related biomarkers by Lactobacillus sakei CJLS03 in a high-fat diet-induced obese murine model.

Recent progresses in clinical diagnostic analyses have demonstrated the decisive influence of host gut microbiota on the status of metabolic disorders. Short chain fatty acids (SCFAs) produced by gut microbiota, in particular, are considered as a key biomarker, both of communication between gut microbiota and the host, and of impact on host metabolic homeostasis. Microbiota modulation and concomitant anti-obesity effects of probiotics have been reported by different researchers. However, the underlying modulatory functions of probiotics on gut microbiota towards host metabolic homeostasis are still not fully understood. In this study, the impact of Lactobacillus sakei CJLS03 (isolated from Korean kimchi) on obesity-related biomarkers was investigated using a diet-induced obese mouse model. Body weight increase, SCFAs, the gut microbiota and various obesity-associated biomarkers were significantly and beneficially influenced by L. sakei CJLS03 administration compared to the control groups. Analytical data on faecal samples support the role of the colonic microbial population in SCFA production. The composition of the latter may be influenced by modulation of the distal gastro-intestinal microbiota by putative probiotics such as L. sakei CJLS03.

Sepsis-Like Systemic Inflammation Induced by Nano-Sized Extracellular Vesicles From Feces.

Nano-sized extracellular vesicles (EVs), including exosomes, microvesicles, and other types of vesicles, are released by most mammalian cells and bacteria. We here ask whether feces contain EVs of mammalian and/or bacterial origin, and whether these EVs induce systemic inflammation. Fecal extracellular vesicles (fEVs) were isolated from mice and humans. The presence of EVs from Gram-negative and Gram-positive bacteria was detected by enzyme-linked immunosorbent assay using anti-lipid A and anti-lipoteichoic acid antibodies, whereas Western blot using anti-beta-actin antibody was employed to detect host-derived EVs in the fEVs. Further, fEVs were administered into mice by intraperitoneal injection, and inflammatory responses were investigated in the peritoneum, blood, and lungs. The role of TLR2 and TLR4 were studied using knockout mice. Significant quantities of EVs were present in feces from mice as well as humans, and derived from Gram-negative and Gram-positive bacteria, as well as the host. Bacteria-free fEVs introduced into the peritoneum induced local and systemic inflammation (including in the lungs), but fEVs from germ-free animals had weaker effects. This pronounced local and systemic inflammatory responses seemed to be induced by EVs from both Gram-negative and Gram-positive bacteria, and was attenuated in mice lacking TLR2 or TLR4. Our findings show that fEVs cause sepsis-like systemic inflammation, when introduced intraperitoneally, a process regulated by TLR2 and TLR4.

Amodiaquine improves insulin resistance and lipid metabolism in diabetic model mice.

AIMS: Although peroxisome proliferator-activated receptors (PPARs)α/γ dual agonists can be beneficial for treatment of dyslipidemia in patients with type 2 diabetes, their use is limited owing to various side effects, including body weight gain, edema, and heart failure. We aimed to demonstrate that amodiaquine, an antimalarial agent, has potential as a PPARα/γ dual agonist with low risk of adverse effects. METHODS: We screened a Prestwick library (Prestwick Chemical; Illkirch, France) to identify novel PPARα/γ dual agonists and selected amodiaquine (4-[(7-chloroquinolin-4-yl)amino]-2-[(diethylamino)methyl]phenol), which activated both PPAR-α & -γ, for further investigation. We performed both in vitro, including glucose uptake assay and fatty acid oxidation assay, and in vivo studies to elucidate the anti-diabetic and anti-obesity effects of amodiaquine. RESULTS: Amodiaquine selectively activated the transcriptional activities of PPARα/γ and enhanced both fatty acid oxidation and glucose uptake without altering insulin secretion in vitro. In high-fat diet-induced obese and genetically modified obese/diabetic mice, amodiaquine not only remarkably ameliorated insulin resistance, hyperlipidemia, and fatty liver but also decreased body weight gain. CONCLUSION: Our findings suggest that amodiaquine exerts beneficial effects on glucose and lipid metabolism by concurrent activation of PPARα/γ. Furthermore, amodiaquine acts as an alternative insulin-sensitizing agent with a positive influence on lipid metabolism and has potential to prevent and treat type 2 diabetes while reducing the risk of lipid abnormalities.

A guanidine-appended scyllo-inositol derivative AAD-66 enhances brain delivery and ameliorates Alzheimer's phenotypes.

Alzheimer's disease (AD) is a degenerative brain disease that destroys memory and other important mental functions but lacks efficient therapeutic agents. Blocking toxic amyloid ß (Aß) could be beneficial for AD and represents a promising therapeutic strategy for AD treatment. scyllo-Inositol (SI) is a potential therapeutic for AD by directly interacting with the Aß peptide to inhibit Aß42 fiber formation. Clinical studies of SI showed promising benefits on mild to moderate AD, however, with limitations on dosage regime. A new strategy to enhance the brain delivery of SI is needed to achieve the efficacy with minimum adverse effects. Herein, we report that a novel guanidine-appended SI derivative AAD-66 resulted in more effective reductions of brain Aß and plaque deposits, gliosis, and behavioral memory deficits in the disease-established 5xFAD mice. Overall, our present study reveals the potential of AAD-66 as a promising therapeutic agent for AD.

Myricetin improves endurance capacity and mitochondrial density by activating SIRT1 and PGC-1α.

Robust mitochondrial respiration provides energy to support physical performance and physiological well-being, whereas mitochondrial malfunction is associated with various pathologies and reduced longevity. In the current study, we tested whether myricetin, a natural flavonol with diverse biological activities, may impact mitochondrial function and longevity. The mice were orally administered myricetin (50 mg/kg/day) for 3 weeks. Myricetin significantly potentiated aerobic capacity in mice, as evidenced by their increased running time and distance. The elevated mitochondrial function was associated with induction of genes for oxidative phosphorylation and mitochondrial biogenesis in metabolically active tissues. Importantly, myricetin treatment led to decreased PGC-1α acetylation through SIRT1 activation. Furthermore, myricetin significantly improved the healthspan and lifespan of wild-type, but not Sir-2.1-deficient, C. elegans. These results demonstrate that myricetin enhances mitochondrial activity, possibly by activating PGC-1α and SIRT1, to improve physical endurance, strongly suggesting myricetin as a mitochondria-activating agent.

Specificity and putative mode of action of a mosquito larvicidal toxin from the bacterium Xenorhabdus innexi.

Reduction of mosquito-borne diseases relies, in part, on the use of synthetic pesticides to control pest mosquitoes. This reliance has led to genetic resistance, environmental contamination and the nondiscriminatory elimination of both pest and non-pest species. To expand our options for control, we screened entomopathogenic bacteria for potential larvicidal activity. A lipopeptide from the bacterium, Xenorhabdus innexi, was discovered that displayed potent larvicidal activity. The LC50s of the lipopeptide towards Aedes aegypti, Culex pipiens and Anopheles gambiae larvae were 1.81, 1.25 and 1.86 parts-per-million, respectively. No mortality was observed in other insect species tested. The putative mode of action of the lipopeptide suggested that after orally ingestion, it bound to the apical membrane of anterior midgut cells and created pores in the cellular membranes. The rapid neutralization of midgut pH suggested the pores disabled the H+-V-ATPase on the basal membrane and led to epithelial cell death. Specificity and toxicity towards mosquito larvae and the unique mode of action makes this lipopeptide a potentially attractive bacterial insecticide for control of mosquitoes.

Lactobacillus plantarum HAC01 regulates gut microbiota and adipose tissue accumulation in a diet-induced obesity murine model.

The functional features of Lactobacillus plantarum HAC01 (HAC01), isolated from fermented Korean kimchi, were studied with regard to the fat mass, immunometabolic biomarkers and dysbiosis in a diet-induced obesity (DIO) murine model. L. rhamnosus GG (LGG) served as reference strain and a PBS-treated group as control. The administration of L. plantarum HAC01 resulted in reduction of the mesenteric adipose depot, the conjunctive tissue closely associated with the gastrointestinal tract, where lipid oxidative gene expression was upregulated compared to the control group. Metagenome analysis of intestinal microbiota showed that both strains HAC01 and LGG influenced specific bacterial families such as the Lachnospiraceae and Ruminococcaceae rather than the phyla Firmicutes and Bacteroidetes as a whole. The relative abundance of the Lachnospiraceae (phylum Firmicutes) was significantly higher in both LAB-treated groups than in the control. Comparing the impact of the two Lactobacillus strains on microbial composition in the gut also suggests strain-specific effects. The study emphasises the need for deeper studies into functional specificity of a probiotic organism at the strain level. Alleviation of obesity-associated dysbiosis by modulation of the gut microbiota appears to be associated with "indicator" bacterial taxa such as the family Lachnospiraceae. This may provide further insight into mechanisms basic to the mode of probiotic action against obesity and associated dysbiosis.

Hyaluronate-Death Receptor 5 Antibody Conjugates for Targeted Treatment of Liver Metastasis.

The liver is the most frequent site of metastasis with a 5-year survival rate of only 20-40%. In this work, hyaluronate (HA)-death receptor 5 antibody (DR5 Ab) conjugate was synthesized as a dual targeting therapeutic agent to treat liver metastasis. Dual targeting was achieved by DR5 Ab, a humanized agonistic monoclonal antibody binding to DR5 frequently overexpressed in many kinds of cancer cells, and by HA, a natural polysaccharide binding to HA receptors highly expressed in both the liver and cancer cells. Thiol end-modified HA was site-specifically conjugated to N-glycan on Fc region of oxidized DR5 Ab using a heterobifunctional linker of 3-(2-pyridyldithio)propionyl hydrazide (PDPH). The successful synthesis of HA-DR5 Ab conjugate was confirmed by (1)H NMR, purpald assay, dynamic light scattering (DLS), and high-performance liquid chromatography (HPLC). In vitro analysis of HA-DR5 Ab conjugate revealed that the conjugation of HA to DR5 Ab did not affect the binding affinity and anticancer efficacy of DR5 Ab. Remarkably, according to in vivo bioimaging study, HA-DR5 Ab conjugate appeared to be highly accumulated in the liver and dramatically effective in inhibiting the tumor growth in liver metastasis model mice.

Standardized Boesenbergia pandurata Extract Stimulates Exercise Endurance Through Increasing Mitochondrial Biogenesis.

In the present study, the effect of standardized Boesenbergia pandurata (Roxb.) Schltr. (fingerroot) ethanol extract on exercise endurance was investigated in L6 rat skeletal muscle cells and C57BL/6J mice. Standardized B. pandurata ethanol extract (BPE) increased mitochondrial mass and stimulated the mRNA expression of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) in vitro. BPE also elevated the mRNA expression of key factors of mitochondrial biogenesis and function, which are activated by PGC-1α, such as estrogen-related receptor α (ERRα), nuclear respiratory factor 1 (NRF-1), and mitochondrial transcription factor A (Tfam). In animal models, both normal and high-fat diet (HFD)-induced obese mice treated with BPE ran much longer than their respective controls. In addition, BPE increased the protein expressions of phosphorylated AMP-activated protein kinase (AMPK), sirtuin 1 (SIRT1), PGC-1α, and peroxisome proliferator-activated receptor delta (PPARδ), which are stimulated by exercise. These results indicate that B. pandurata could be a potential nutraceutical candidate for enhancing exercise endurance based on its mitochondrial biogenesis and exercise-mimicking effects.

A Fomitopsis pinicola Jeseng Formulation Has an Antiobesity Effect and Protects against Hepatic Steatosis in Mice with High-Fat Diet-Induced Obesity.

This study investigated the antiobesity effect of an extract of the Fomitopsis pinicola Jeseng-containing formulation (FAVA), which is a combination of four natural components: Fomitopsis pinicola Jeseng; Acanthopanax senticosus; Viscum album coloratum; and Allium tuberosum. High-fat diet- (HFD-) fed male C57BL/6J mice were treated with FAVA (200 mg/kg/day) for 12 weeks to monitor the antiobesity effect and amelioration of nonalcoholic fatty liver diseases (NAFLD). Body and white adipose tissue (WAT) weights were reduced in FAVA-treated mice, and a histological examination showed an amelioration of fatty liver in FAVA-treated mice without decreasing food consumption. Additionally, FAVA reduced serum lipid profiles, leptin, and insulin levels compared with the HFD control group. The FAVA extract suppressed lipogenic mRNA expression levels from WAT concomitantly with the cholesterol biosynthesis level in the liver. These results demonstrate the inhibitory effects of FAVA on obesity and NAFLD in the diet-induced obese (DIO) mouse model. Therefore, FAVA may be an effective therapeutic candidate for treating obesity and fatty liver caused by a high-fat diet.

Daphnane Diterpenes from Daphne genkwa Activate Nurr1 and Have a Neuroprotective Effect in an Animal Model of Parkinson's Disease.

Nurr1 is an orphan nuclear receptor that is essential for the differentiation and maintenance of dopaminergic neurons in the brain, and it is a therapeutic target for Parkinson's disease (PD). During the screening for Nurr1 activators from natural sources using cell-based assay systems, a methanol extract of the combined stems and roots of Daphne genkwa was found to activate the transcriptional function of Nurr1 at a concentration of 3 µg/mL. The active components were isolated and identified as genkwanine N (1) and yuanhuacin (2). Both compounds 1 and 2 significantly enhanced the function of Nurr1 at 0.3 µM. Nurr1-specific siRNA abolished the activity of 1 and 2, strongly suggesting that transcriptional activation by 1 and 2 occurred through the modulation of Nurr1 function. Additionally, treatment with 1 and 2 inhibited 6-hydroxydopamine (6-OHDA)-induced neuronal cell death and lipopolysaccharide (LPS)-induced neuroinflammation. Moreover, in a 6-OHDA-lesioned rat model of PD, intraperitoneal administration of 2 (0.5 mg/kg/day) for 2 weeks significantly improved behavioral deficits and reduced tyrosine hydroxylase (TH)-positive dopaminergic neuron death induced by 6-OHDA injection and had a beneficial effect on the inflammatory response in the brain. Accordingly, compounds 1 and 2, the first reported Nurr1 activators of natural origin, are potential lead compounds for the treatment of PD.

Vaccinia-related kinase 1 promotes hepatocellular carcinoma by controlling the levels of cell cycle regulators associated with G1/S transition.

We identified the specific role of vaccinia-related kinase 1 (VRK1) in the progression of hepatocellular carcinoma (HCC) and evaluated its therapeutic and prognostic potential. VRK1 levels were significantly higher in HCC cell lines than a normal hepatic cell line, and were higher in HCC than non-tumor tissue. VRK1 knockdown inhibited the proliferation of SK-Hep1, SH-J1 and Hep3B cells; moreover, depletion of VRK1 suppressed HCC tumor growth in vivo. We also showed that VRK1 knockdown increased the number of G1 arrested cells by decreasing cyclin D1 and p-Rb while upregulating p21 and p27, and that VRK1 depletion downregulated phosphorylation of CREB, a transcription factor regulating CCND1. Additionally, we found that luteolin, a VRK1 inhibitor, suppressed HCC growth in vitro and in vivo, and that the aberrant VRK1 expression correlated with poor prognostic features of HCC. High levels of VRK1 were associated with shorter overall and disease-free survival and higher recurrence rates. Taken together, our findings suggest VRK1 may act as a tumor promoter by controlling the level of cell cycle regulators associated with G1/S transition and could potentially serve as a therapeutic target and/or prognostic biomarker for HCC.

Ursolic acid exerts anti-cancer activity by suppressing vaccinia-related kinase 1-mediated damage repair in lung cancer cells.

Many mitotic kinases have been targeted for the development of anti-cancer drugs, and inhibitors of these kinases have been expected to perform well for cancer therapy. Efforts focused on selecting good targets and finding specific drugs to target are especially needed, largely due to the increased frequency of anti-cancer drugs used in the treatment of lung cancer. Vaccinia-related kinase 1 (VRK1) is a master regulator in lung adenocarcinoma and is considered a key molecule in the adaptive pathway, which mainly controls cell survival. We found that ursolic acid (UA) inhibits the catalytic activity of VRK1 via direct binding to the catalytic domain of VRK1. UA weakens surveillance mechanisms by blocking 53BP1 foci formation induced by VRK1 in lung cancer cells, and possesses synergistic anti-cancer effects with DNA damaging drugs. Taken together, UA can be a good anti-cancer agent for targeted therapy or combination therapy with DNA damaging drugs for lung cancer patients.

Deficiency of Capicua disrupts bile acid homeostasis.

Capicua (CIC) has been implicated in pathogenesis of spinocerebellar ataxia type 1 and cancer in mammals; however, the in vivo physiological functions of CIC remain largely unknown. Here we show that Cic hypomorphic (Cic-L(-/-)) mice have impaired bile acid (BA) homeostasis associated with induction of proinflammatory cytokines. We discovered that several drug metabolism and BA transporter genes were down-regulated in Cic-L(-/-) liver, and that BA was increased in the liver and serum whereas bile was decreased within the gallbladder of Cic-L(-/-) mice. We also found that levels of proinflammatory cytokine genes were up-regulated in Cic-L(-/-) liver. Consistent with this finding, levels of hepatic transcriptional regulators, such as hepatic nuclear factor 1 alpha (HNF1α), CCAAT/enhancer-binding protein beta (C/EBPß), forkhead box protein A2 (FOXA2), and retinoid X receptor alpha (RXRα), were markedly decreased in Cic-L(-/-) mice. Moreover, induction of tumor necrosis factor alpha (Tnfα) expression and decrease in the levels of FOXA2, C/EBPß, and RXRα were found in Cic-L(-/-) liver before BA was accumulated, suggesting that inflammation might be the cause for the cholestasis in Cic-L(-/-) mice. Our findings indicate that CIC is a critical regulator of BA homeostasis, and that its dysfunction might be associated with chronic liver disease and metabolic disorders.