Hepatocyte activity of the cholesterol sensor smoothened regulates cholesterol and bile acid homeostasis in mice.

Cellular cholesterol is regulated by at least two transcriptional mechanisms involving sterol-regulatory-element-binding proteins (SREBPs) and liver X receptors (LXRs). Although SREBP and LXR pathways are the predominant mechanisms that sense cholesterol in the endoplasmic reticulum and nucleus to alter sterol-regulated gene expression, evidence suggests cholesterol in plasma membrane can be sensed by proteins in the Hedgehog (Hh) pathway which regulate organ self-renewal and are a morphogenic driver during embryonic development. Cholesterol interacts with the G-protein-coupled receptor Smoothened (Smo), which impacts downstream Hh signaling. Although evidence suggests cholesterol influences Hh signaling, it is not known whether Smo-dependent sterol sensing impacts cholesterol homeostasis in vivo. We examined dietary-cholesterol-induced reorganization of whole-body sterol and bile acid (BA) homeostasis in adult mice with inducible hepatocyte-specific Smo deletion. These studies demonstrate Smo in hepatocytes plays a regulatory role in sensing and feedback regulation of cholesterol balance driven by excess dietary cholesterol.

Pitcher pot neourethral modification of ileal orthotopic neobladder achieves satisfactory long-term functional and quality of life outcomes with low clean intermittent self-catheterization rate.

Objective: To describe a decade of our experience with a neo-urethral modification of ileal orthotopic neobladder (pitcher pot ONB). Multiple investigators have reported similar modifications. However, long-term longitudinal functional and quality of life (QOL) outcomes are lacking. Methods: Prospectively maintained hospital registry for 238 ONB patients comprising a mix of open and robotic surgery cohorts from 2007 to 2017, and minimum of 2 years of follow-up was retrospectively queried. QOL was evaluated using Bladder Cancer Index (BCI). Longitudinal trends of QOL domain parameters were analysed. List of perioperative variables that have a biologically plausible association with continence, potency, and post-operative BCI QOL sexual, urinary, and bowel domain scores was drawn. Variables included surgery type, Body Mass Index (BMI), T and N stage, neurovascular bundle (NVB) sparing, age, and related pre-operative BCI QOL domain score. Prognostic associations were analysed using multivariable Cox proportional hazard models and multilevel mixed-effects modeling. Results: The study comprised 80 and 158 patients who underwent open and robotic sandwich technique cohorts, respectively. Open surgery was associated with significantly higher "any" complication (40% vs 27%, P-value .050) and "major" complication rate (15% vs 11%, P-value .048). All patients developed a bladder capacity >400 cc with negligible post-void residual urine, and all but one patient achieved spontaneous voiding by the end of study period (<1% clean intermittent self-catheterization [CISC] rate). By 15 months, QOL for all three domains had recovered to reach a plateau. About 45% of patients achieved potency, and the median time to achieve day and night time continence was 9 and 12 months respectively. Lower age and NVBs spared during surgery were found to be significantly associated with the earlier achievement of potency, day and night time continence, as well as better urinary and sexual summary QOL scores. Conclusions: Pitcher pot neobladder achieves satisfactory long-term functional and QOL outcomes with negligible CISC rate. Results were superior with incremental nerves spared during surgery.

Small molecule inhibition of gut microbial choline trimethylamine lyase activity alters host cholesterol and bile acid metabolism.

The gut microbe-derived metabolite trimethylamine-N-oxide (TMAO) has recently been linked to cardiovascular disease (CVD) pathogenesis, prompting the development of therapeutic strategies to reduce TMAO. Previous work has shown that experimental alteration of circulating TMAO levels via dietary alterations or inhibition of the host TMAO producing enzyme flavin containing monooxygenase 3 (FMO3) is associated with reorganization of host cholesterol and bile acid metabolism in mice. In this work, we set out to understand whether recently developed nonlethal gut microbe-targeting small molecule choline trimethylamine (TMA) lyase inhibitors also alter host cholesterol and bile acid metabolism. Treatment of mice with the mechanism-based choline TMA lyase inhibitor, iodomethylcholine (IMC), increased fecal neutral sterol loss in the form of coprostanol, a bacteria metabolite of cholesterol. In parallel, IMC treatment resulted in marked reductions in the intestinal sterol transporter Niemann-pick C1-like 1 (NPC1L1) and reorganization of the gut microbial community, primarily reversing choline supplemented diet-induced changes. IMC also prevented diet-driven hepatic cholesterol accumulation, causing both upregulation of the host hepatic bile acid synthetic enzyme CYP7A1 and altering the expression of hepatic genes critical for bile acid feedback regulation. These studies suggest that the gut microbiota-driven TMAO pathway is closely linked to both microbe and host sterol and bile acid metabolism. Collectively, as gut microbe-targeting choline TMA lyase inhibitors move through the drug discovery pipeline from preclinical models to human studies, it will be important to understand how these drugs impact both microbe and host cholesterol and bile acid metabolism.NEW & NOTEWORTHY The gut microbe-dependent metabolite trimethylamine-N-oxide (TMAO) has been strongly associated with cardiovascular mortality, prompting drug discovery efforts to identify points of therapeutic intervention within the microbe host TMAO pathway. Recently, mechanism-based small molecule inhibitors of the major bacterial trimethylamine (TMA) lyase enzymes have been developed, and these drugs show efficacy as anti-atherothrombotic agents. The novel findings of this study are that small molecule TMA lyase inhibition results in beneficial reorganization of host cholesterol and bile acid metabolism. This study confirms previous observations that the gut microbial TMAO pathway is intimately linked to host cholesterol and bile acid metabolism and provides further rationale for the development of small molecule choline TMA lyase inhibitors for the treatment of cardiometabolic disorders.

Adjuvant radiation compares favorably to chemotherapy in patients with carcinoma penis and nodal positivity restricted to groin.

OBJECTIVES: To compare overall survival (OS) between adjuvant radiation, chemotherapy and chemoradiation (CCRT) postsurgery for node-positive patients with carcinoma penis. METHODS: Prospectively maintained registry for 45 patients receiving adjuvant treatment following lymph node dissection from 2011 to 2017, having minimum 6 months follow-up and more than 2 positive inguinal nodes was analyzed. Patients without pelvic nodal positivity (n= 32) were treated by radiotherapy (RT) (n = 25) or chemotherapy (n = 7); CCRT (n = 6) or chemotherapy (n = 7) was used in patients with positive pelvic nodes (n = 13). Data was collected for age, comorbidities, body mass index, tobacco exposure, treatment modality, tumor grade, pathological T and N stage, and extra-nodal extension. OS was compared between different treatment modalities stratifying patients with and without pelvic nodal positivity. Multivariate cox proportional hazard analysis was used to narrow down remaining variables and Inverse Probability Treatment Weights modeling was used to determine average treatment effect. RESULTS: About 12 of 14 patients in the chemotherapy group received both cisplatin and paclitaxel. Pathological T stage, N stage and extra-nodal extension had significant association with OS on multivariate analysis. Among patients with nodal positivity restricted to groin the estimated average OS when all patients received adjuvant RT was 1,438 days (95% confidence interval [CI] 1,256-1,619 days, Pvalue <0.0001). The estimated average OS if all patients received chemotherapy was lower by 1,007 days (95% CI 810-1,202 days, P value <0.0001). Among patients with positive pelvic nodes the estimated average OS when all patients received adjuvant CCRT was 467 days (95% CI 368-566 days, P value <0.0001). The estimated average OS difference if all patients received chemotherapy was 17 days (95% CI -144 to 178 days, Pvalue 0.21). CONCLUSION: In patients with nodal positivity limited to groin, adjuvant RT proved superior to chemotherapy. Among patients with pelvic nodal positivity, CCRT offers no significant OS advantage over combination chemotherapy.

Obesity-linked suppression of membrane-bound O-acyltransferase 7 (MBOAT7) drives non-alcoholic fatty liver disease.

Recent studies have identified a genetic variant rs641738 near two genes encoding membrane bound O-acyltransferase domain-containing 7 (MBOAT7) and transmembrane channel-like 4 (TMC4) that associate with increased risk of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcohol-related cirrhosis, and liver fibrosis in those infected with viral hepatitis (Buch et al., 2015; Mancina et al., 2016; Luukkonen et al., 2016; Thabet et al., 2016; Viitasalo et al., 2016; Krawczyk et al., 2017; Thabet et al., 2017). Based on hepatic expression quantitative trait loci analysis, it has been suggested that MBOAT7 loss of function promotes liver disease progression (Buch et al., 2015; Mancina et al., 2016; Luukkonen et al., 2016; Thabet et al., 2016; Viitasalo et al., 2016; Krawczyk et al., 2017; Thabet et al., 2017), but this has never been formally tested. Here we show that Mboat7 loss, but not Tmc4, in mice is sufficient to promote the progression of NAFLD in the setting of high fat diet. Mboat7 loss of function is associated with accumulation of its substrate lysophosphatidylinositol (LPI) lipids, and direct administration of LPI promotes hepatic inflammatory and fibrotic transcriptional changes in an Mboat7-dependent manner. These studies reveal a novel role for MBOAT7-driven acylation of LPI lipids in suppressing the progression of NAFLD.

Sterol 12α-Hydroxylase Aggravates Dyslipidemia by Activating the Ceramide/mTORC1/SREBP-1C Pathway via FGF21 and FGF15.

Sterol 12α-hydroxylase (CYP8B1) is required for the synthesis of cholic acid in the classic bile acid synthesis pathway and plays a role in dyslipidemia and insulin resistance. However, the mechanism of the involvement of Cyp8b1 in dyslipidemia and insulin resistance is not known. CYP8B1 mRNA and protein expression are elevated in diabetic and obese (db/db) mouse liver. In this study adenovirus-mediated transduction of CYP8B1 was used to study the effect of Cyp8b1 on lipid metabolism in mice. Results show that Ad-Cyp8b1 increased 12α-hydroxylated bile acids and induced sterol regulatory element-binding protein 1c (Srebp-1c)-mediated lipogenic gene expression. Interestingly, Ad-Cyp8b1 increased ceramide synthesis and activated hepatic mechanistic target of rapamycin complex 1 (mTORC1)-p70S6K signaling cascade and inhibited AKT/insulin signaling in mice. Ad-Cyp8b1 increased free fatty acid uptake into mouse primary hepatocytes. Ceramides stimulated S6K phosphorylation in both mouse and human primary hepatocytes. In high-fat diet-fed mice, Ad-Cyp8b1 reduced fibroblast growth factor 21 (FGF21), activated intestinal farnesoid X receptor (FXR) target gene expression, increased serum ceramides, VLDL secretion, and LDL cholesterol. In high-fat diet-induced obese (DIO) mice, Cyp8b1 ablation by adenovirus-mediated shRNA improved oral glucose tolerance, increased FGF21, and reduced liver triglycerides, inflammatory cytokine expression, nuclear localization of Srebp-1c and phosphorylation of S6K. In conclusion, this study unveiled a novel mechanism linking CYP8B1 to ceramide synthesis and mTORC1 signaling in dyslipidemia and insulin resistance, via intestinal FXR-mediated induction of FGF15 and liver FGF21. Reducing cholic acid synthesis may be a potential therapeutic strategy to treat dyslipidemia and nonalcoholic fatty liver disease.

Deficiency of Both Farnesoid X Receptor and Takeda G Protein-Coupled Receptor 5 Exacerbated Liver Fibrosis in Mice.

Activation of the nuclear bile acid receptor farnesoid X receptor (FXR) protects against hepatic inflammation and injury, while Takeda G protein-coupled receptor 5 (TGR5) promotes adipose tissue browning and energy metabolism. Here, we examined the physiological and metabolic effects of the deficiency of these two bile acid receptors on hepatic metabolism and injury in mice. Fxr/Tgr5 double knockout mice (DKO) were generated for metabolic phenotyping. Male DKO mice fed a chow diet had reduced liver lipid levels but increased serum cholesterol levels. Liver cholesterol 7α-hydroxylase (Cyp7a1) activity and sterol 12α-hydroxylase mRNA levels were induced, while ileum FXR target genes were suppressed in DKO mice compared to wild-type (WT) mice. Bile acid pool size was increased in DKO mice, with increased taurocholic acid and decreased tauromuricholic acids. RNA sequencing analysis of the liver transcriptome revealed that bile acid synthesis and fibrosis gene expression levels are increased in chow-fed DKO mice compared to WT mice and that the top regulated pathways are involved in steroid/cholesterol biosynthesis, liver cirrhosis, and connective tissue disease. Cholestyramine treatment further induced Cyp7a1 mRNA and protein in DKO mice and increased bile acid pool size, while cholic acid also induced Cyp7a1 in DKO mice, suggesting impaired bile acid feedback regulation. A Western diet containing 0.2% cholesterol increased oxidative stress and markers of liver fibrosis but not hepatic steatosis in DKO mice. Conclusion: FXR and TGR5 play critical roles in protecting the liver from inflammation and fibrosis, and deficiency of both of these bile acid receptors in mice increased cholic acid synthesis and the bile acid pool, liver fibrosis, and inflammation; FXR and TGR5 DKO mice may be a model for liver fibrosis.

KLF15 regulates endobiotic and xenobiotic metabolism.

Hepatic metabolism and elimination of endobiotics (for example, steroids, bile acids) and xenobiotics (for example, drugs, toxins) is essential for health. While the enzymatic (termed phase I-II) and transport machinery (termed phase III) controlling endobiotic and xenobiotic metabolism (EXM) is known, understanding of molecular nodal points that coordinate EXM function in physiology and disease remains incomplete. Here we show that the transcription factor Kruppel-like factor 15 (KLF15) regulates all three phases of the EXM system by direct and indirect pathways. Unbiased transcriptomic analyses coupled with validation studies in cells, human tissues, and animals, support direct transcriptional control of the EXM machinery by KLF15. Liver-specific deficiency of KLF15 (Li-KO) results in altered expression of numerous phase I-III targets, and renders animals resistant to the pathologic effects of bile acid and acetaminophen toxicity. Furthermore, Li-KO mice demonstrate enhanced degradation and elimination of endogenous steroid hormones, such as testosterone and glucocorticoid, resulting in reduced male fertility and blood glucose levels, respectively. Viral reconstitution of hepatic KLF15 expression in Li-KO mice reverses these phenotypes. Our observations identify a previously unappreciated transcriptional pathway regulating metabolism and elimination of endobiotics and xenobiotics.

Simultaneous integrated boost by Intensity Modulated Radiotherapy (SIB-IMRT) in patients undergoing breast conserving surgery - A clinical and dosimetric perspective.

AIM: To study feasibility of simultaneous integrated boost by intensity modulated radiotherapy (SIB-IMRT) in patients undergoing breast conserving surgery and its impact on cosmesis and dosimetry. PATIENTS AND METHODS: Between January 2014 and June 2017, all breast cancer patients fulfilling inclusion and exclusion criteria were enrolled in a prospective study conducted at a tertiary cancer centre in North India. All patients received adjuvant radiotherapy by simultaneous integrated boost technique following breast conserving surgery. Clinical information including patient and pathological characteristics, observed acute and chronic toxicities along with cosmesis using Harvard score were recorded and analysed. Univariate analysis and multivariate logistic regression analysis were performed for those variables which were found to be significant (p < 0.050) to study the influence of clinicopathological and dosimetric factors on toxicity and cosmetic outcome. RESULTS: Maximum acute skin toxicity during treatment was Grade 0-1 in 68.2% and Grade 2-3 in 31.8% of cases, respectively. Fibrosis was the commonest late toxicity with ≥Grade II fibrosis being noted in 16.3% of cases. Assessment of global cosmesis at 12 months follow-up showed good/excellent cosmesis in 88.4% of cases. Mean age, tumor size and homogeneity index (HI) were the significant factors associated with fair or poor cosmetic outcome and ≥Grade 2 fibrosis on multivariate analysis. Telengectasia and breast edema were more frequent in patients with larger tumor size/GTV volume. There were 5 recurrences including 1 ipsilateral local breast tumor recurrence. CONCLUSION: SIB-IMRT is a dosimetrically feasible option in patients undergoing breast conserving surgery and provides good/excellent cosmetic outcome.

Intestine farnesoid X receptor agonist and the gut microbiota activate G-protein bile acid receptor-1 signaling to improve metabolism.

Bile acids activate farnesoid X receptor (FXR) and G protein-coupled bile acid receptor-1 (aka Takeda G protein-coupled receptor-5 [TGR5]) to regulate bile acid metabolism and glucose and insulin sensitivity. FXR and TGR5 are coexpressed in the enteroendocrine L cells, but their roles in integrated regulation of metabolism are not completely understood. We reported recently that activation of FXR induces TGR5 to stimulate glucagon-like peptide-1 (GLP-1) secretion to improve insulin sensitivity and hepatic metabolism. In this study, we used the intestine-restricted FXR agonist fexaramine (FEX) to study the effect of activation of intestinal FXR on the gut microbiome, bile acid metabolism, and FXR and TGR5 signaling. The current study revealed that FEX markedly increased taurolithocholic acid, increased secretion of fibroblast growth factors 15 and 21 and GLP-1, improved insulin and glucose tolerance, and promoted white adipose tissue browning in mice. Analysis of 16S ribosomal RNA sequences of the gut microbiome identified the FEX-induced and lithocholic acid-producing bacteria Acetatifactor and Bacteroides. Antibiotic treatment completely reversed the FEX-induced metabolic phenotypes and inhibited taurolithocholic acid synthesis, adipose tissue browning, and liver bile acid synthesis gene expression but further increased intestinal FXR target gene expression. FEX treatment effectively improved lipid profiles, increased GLP-1 secretion, improved glucose and insulin tolerance, and promoted adipose tissue browning, while antibiotic treatment reversed the beneficial metabolic effects of FEX in obese and diabetic mice. CONCLUSION: This study uncovered a mechanism in which activation of intestinal FXR shaped the gut microbiota to activate TGR5/GLP-1 signaling to improve hepatic glucose and insulin sensitivity and increase adipose tissue browning; the gut microbiota plays a critical role in bile acid metabolism and signaling to regulate metabolic homeostasis in health and disease. (Hepatology 2018).

Farnesoid X receptor induces Takeda G-protein receptor 5 cross-talk to regulate bile acid synthesis and hepatic metabolism.

The bile acid-activated receptors, nuclear farnesoid X receptor (FXR) and the membrane Takeda G-protein receptor 5 (TGR5), are known to improve glucose and insulin sensitivity in obese and diabetic mice. However, the metabolic roles of these two receptors and the underlying mechanisms are incompletely understood. Here, we studied the effects of the dual FXR and TGR5 agonist INT-767 on hepatic bile acid synthesis and intestinal secretion of glucagon-like peptide-1 (GLP-1) in wild-type, Fxr-/-, and Tgr5-/- mice. INT-767 efficaciously stimulated intracellular Ca2+ levels, cAMP activity, and GLP-1 secretion and improved glucose and lipid metabolism more than did the FXR-selective obeticholic acid and TGR5-selective INT-777 agonists. Interestingly, INT-767 reduced expression of the genes in the classic bile acid synthesis pathway but induced those in the alternative pathway, which is consistent with decreased taurocholic acid and increased tauromuricholic acids in bile. Furthermore, FXR activation induced expression of FXR target genes, including fibroblast growth factor 15, and unexpectedly Tgr5 and prohormone convertase 1/3 gene expression in the ileum. We identified an FXR-responsive element on the Tgr5 gene promoter. Fxr-/- and Tgr5-/- mice exhibited reduced GLP-1 secretion, which was stimulated by INT-767 in the Tgr5-/- mice but not in the Fxr-/- mice. Our findings uncovered a novel mechanism in which INT-767 activation of FXR induces Tgr5 gene expression and increases Ca2+ levels and cAMP activity to stimulate GLP-1 secretion and improve hepatic glucose and lipid metabolism in high-fat diet-induced obese mice. Activation of both FXR and TGR5 may therefore represent an effective therapy for managing hepatic steatosis, obesity, and diabetes.

Intestinal Farnesoid X Receptor and Takeda G Protein Couple Receptor 5 Signaling in Metabolic Regulation.

Bile acids play a critical role in the regulation of glucose, lipid and energy metabolisms by activating the nuclear bile acid receptor farnesoid X receptor (FXR) and membrane G protein-coupled bile acid receptor-1 (aka takeda G protein couple receptor 5, TGR5) signaling. Paradoxical roles of FXR in the regulation of glucose and lipid metabolism and metabolic disorder have been reported recently. The activation or inhibition of intestinal FXR signaling has been shown to improve insulin and glucose sensitivity and energy metabolism to prevent diabetes, obesity and non-alcoholic fatty liver disease (NAFLD). TGR5 has an anti-inflammatory function in the intestine and stimulates glucagon-like peptide-1 (GLP-1) secretion in the intestine to stimulate insulin secretion from the pancreas. The role of TGR5 in metabolism and metabolic regulation is not clear and warrants further study. FXR and TGR5 are co-expressed in the ileum and colon. These 2 bile acid-activated receptors may cooperate to stimulate GLP-1 secretion and improve hepatic metabolism. FXR and TGR5 dual agonists may have therapeutic potential for treating diabetes and NAFLD.

Cholesterol 7α-hydroxylase protects the liver from inflammation and fibrosis by maintaining cholesterol homeostasis.

Cholesterol 7α-hydroxylase (CYP7A1) plays a critical role in control of bile acid and cholesterol homeostasis. Bile acids activate farnesoid X receptor (FXR) and Takeda G protein-coupled receptor 5 (TGR5) to regulate lipid, glucose, and energy metabolism. However, the role of bile acids in hepatic inflammation and fibrosis remains unclear. In this study, we showed that adenovirus-mediated overexpression of Cyp7a1 ameliorated lipopolysaccharide (LPS)-induced inflammatory cell infiltration and pro-inflammatory cytokine production in WT and TGR5-deficient (Tgr5-/-) mice, but not in FXR-deficient (Fxr-/-) mice, suggesting that bile acid signaling through FXR protects against hepatic inflammation. Nuclear factor κ light-chain enhancer of activated B cells (NF-κB)-luciferase reporter assay showed that FXR agonists significantly inhibited TNF-α-induced NF-κB activity. Furthermore, chromatin immunoprecipitation and mammalian two-hybrid assays showed that ligand-activated FXR interacted with NF-κB and blocked recruitment of steroid receptor coactivator-1 to cytokine promoter and resulted in inhibition of NF-κB activity. Methionine/choline-deficient (MCD) diet increased hepatic inflammation, free cholesterol, oxidative stress, apoptosis, and fibrosis in CYP7A1-deficient (Cyp7a1-/-) mice compared with WT mice. Remarkably, adenovirus-mediated overexpression of Cyp7a1 effectively reduced hepatic free cholesterol and oxidative stress and reversed hepatic inflammation and fibrosis in MCD diet-fed Cyp7a1-/- mice. Current studies suggest that increased Cyp7a1 expression and bile acid synthesis ameliorate hepatic inflammation through activation of FXR, whereas reduced bile acid synthesis aggravates MCD diet-induced hepatic inflammation and fibrosis. Maintaining bile acid and cholesterol homeostasis is important for protecting against liver injury and nonalcoholic fatty liver disease.

Retinoic acid-related orphan receptor α regulates diurnal rhythm and fasting induction of sterol 12α-hydroxylase in bile acid synthesis.

Sterol 12α-hydroxylase (CYP8B1) is required for cholic acid synthesis and plays a critical role in intestinal cholesterol absorption and pathogenesis of cholesterol gallstone, dyslipidemia, and diabetes. In this study we investigated the underlying mechanism of fasting induction and circadian rhythm of CYP8B1 by a cholesterol-activated nuclear receptor and core clock gene retinoic acid-related orphan receptor α (RORα). Fasting stimulated, whereas restricted-feeding reduced expression of CYP8B1 mRNA and protein. However, fasting and feeding had little effect on the diurnal rhythm of RORα mRNA expression, but fasting increased RORα protein levels by cAMP-activated protein kinase A-mediated phosphorylation and stabilization of the protein. Adenovirus-mediated gene transduction of RORα to mice strongly induced CYP8B1 expression, and increased liver cholesterol and 12α-hydroxylated bile acids in the bile acid pool and serum. A reporter assay identified a functional RORα response element in the CYP8B1 promoter. RORα recruited cAMP response element-binding protein-binding protein (CBP) to stimulate histone acetylation on the CYP8B1 gene promoter. In conclusion, RORα is a key regulator of diurnal rhythm and fasting induction of CYP8B1, which regulates bile acid composition and serum and liver cholesterol levels. Antagonizing RORα activity may be a therapeutic strategy for treating inflammatory diseases such as non-alcoholic fatty liver disease and type 2 diabetes.