Sirtuin 1 activation alleviates cholestatic liver injury in a cholic acid-fed mouse model of cholestasis.

Sirtuin1 (Sirt1; mammalian homolog of Saccharomyces cerevisiae enzyme Sir2) is a transcriptional and transactivational regulator of murine farnesoid X receptor (Fxr), which is the primary bile acid (BA) sensor, and critical regulator of BA metabolism in physiological and pathophysiological conditions. Previous studies have suggested compromised Sirt1 expression in rodent models of cholestatic liver injury. We hypothesized that Sirt1 could be potentially targeted to alleviate cholestatic liver injury. In cultured primary human hepatocytes, SIRT1 messenger RNA was down-regulated after GCA treatment, potentially through induction of microRNA (miR)-34a, whereas tauroursodeoxycholic acid induced SIRT1 expression without affecting miR-34a expression. Sirt1 expression was also significantly down-regulated in three mouse models of liver injury (bile duct ligation, 1% cholic acid [CA] fed, and the Mdr2-/- mouse). Mice fed CA diet also demonstrated hepatic FXR hyperacetylation and induction of the Janus kinase/p53 pathway. Mice fed a CA diet and concurrently administered the Sirt1 activator, SRT1720 (50 mg/kg/day, orally), demonstrated 40% and 45% decrease in plasma alanine aminotransferase and BA levels, respectively. SRT1720 increased hepatic BA hydrophilicity by increasing tri- and tetrahydroxylated and decreasing the dihydroxylated BA fraction. SRT1720 administration also inhibited hepatic BA synthesis, potentially through ileal fibroblast growth factor 15- and Fxr-mediated inhibition of cytochrome p450 (Cyp) 7a1 and Cyp27a1, along with increased hepatic BA hydroxylation in association with Cyp2b10 induction. SRT1720 administration significantly induced renal multidrug resistance-associated protein 2 and 4, peroxisome proliferator-activated receptor gamma coactivator 1-α, and constitutive androstance receptor expression along with ∼2-fold increase in urinary BA concentrations. CONCLUSION: SRT1720 administration alleviates cholestatic liver injury in mice by increasing hydrophilicity of hepatic BA composition and decreasing plasma BA concentration through increased BA excretion into urine. Thus, use of small-molecule activators of Sirt1 presents a novel therapeutic target for cholestatic liver injury. (Hepatology 2016;64:2151-2164).

The value of breast MRI in high-risk patients with newly diagnosed breast cancer to exclude invasive disease in the contralateral prophylactic mastectomy: Is there a role to choose wisely patients for sentinel node biopsy?

The aim of this study was to evaluate the presence of clinically and mammographically occult disease using breast MRI in a cohort of cancer patients undergoing contralateral prophylactic mastectomy (CPM) and the utmost indication of axillary assessment (sentinel node biopsy (SLNB)) for this side. A retrospective review of patients with unilateral invasive breast cancer or ductal carcinoma in situ (DCIS) from institutional MRI registry data (2004-2010) was conducted. Characteristics of patients undergoing CPM with breast MRI obtained less than 6 month before surgery were evaluated. A total of 2322 consecutive patients diagnosed with DCIS or stage I to III infiltrating breast cancer underwent preoperative breast MRI. Of these, 1376 patients (59.2%) had contralateral clinical breast exam and mammography without abnormalities; and 116 patients (4.9%) underwent CPM (28 excluded patients had breast MRI more than 6 months before CPM). The mean age of the 88 patients was 49 years (range 28-76 years). Two (2.3%) DCIS identified on surgical pathology specimen were not depicted by MRI and the 5 mm T1N0 invasive cancer (1.1%) was identified on MRI. Preoperative MRI showed 95% accuracy to demonstrate absence of occult disease with negative predicted value (NPV) of 98% (95% CI: 91.64-99.64%). Occult disease was present in 3.4% of CPM. MRI accurately identified the case of invasive cancer in this cohort. The high negative predictive value suggests that MRI can be used to select patients without consideration of SLNB for the contralateral side.

Bisphenol A sulfonation is impaired in metabolic and liver disease.

BACKGROUND: Bisphenol A (BPA) is a widely used industrial chemical and suspected endocrine disruptor to which humans are ubiquitously exposed. The liver metabolizes and facilitates BPA excretion through glucuronidation and sulfonation. The sulfotransferase enzymes contributing to BPA sulfonation (detected in human and rodents) is poorly understood. OBJECTIVES: To determine the impact of metabolic and liver disease on BPA sulfonation in human and mouse livers. METHODS: The capacity for BPA sulfonation was determined in human liver samples that were categorized into different stages of metabolic and liver disease (including obesity, diabetes, steatosis, and cirrhosis) and in livers from ob/ob mice. RESULTS: In human liver tissues, BPA sulfonation was substantially lower in livers from subjects with steatosis (23%), diabetes cirrhosis (16%), and cirrhosis (18%), relative to healthy individuals with non-fatty livers (100%). In livers of obese mice (ob/ob), BPA sulfonation was lower (23%) than in livers from lean wild-type controls (100%). In addition to BPA sulfonation activity, Sult1a1 protein expression decreased by 97% in obese mouse livers. CONCLUSION: Taken together these findings establish a profoundly reduced capacity of BPA elimination via sulfonation in obese or diabetic individuals and in those with fatty or cirrhotic livers versus individuals with healthy livers.

Evaluation of Apparent Diffusion Coefficient to Predict Grade, Microinvasion, and Invasion in Ductal Carcinoma In Situ of the Breast.

RATIONALE AND OBJECTIVES: To evaluate the role of apparent diffusion coefficient (ADC) in distinguishing ductal carcinoma in situ (DCIS) grades and identifying microinvasive and/or invasive disease in the preoperative evaluation of patients with core biopsy-proven DCIS. MATERIALS AND METHODS: Research Ethics Board-approved study with informed consent from 81 women (age, 36-84 years) scheduled for core-biopsy with results of 82 noninvasive breast carcinomas. All patients were assessed preoperatively by diffusion sequence in addition to contrast magnetic resonance imaging (MRI). Lesion morphology and ADC values were recorded. The Kruskal-Wallis or one-way analysis of variance test and Pearson correlation coefficient were used to study the association between ADC and MRI lesion characteristics. Logistic regression analysis was used to evaluate the ability of ADC to predict the presence of invasion. RESULTS: Surgical pathology demonstrated associated invasive cancer in 26.8%, microinvasion in 14.6%, and pure DCIS in 58.5%. The minimum regions of interest (ROI)-based ADC was significantly different among the following three groups (P < .001, Kruskal-Wallis test): 0.98 × 10(-3) mm(2)/s ± 0.25 for pure DCIS, 0.82 × 10(-3) mm(2)/s ± 0.20 for DCIS with microinvasion, and 0.71 × 10(-3) mm(2)/s ± 0.27 for DCIS with invasive disease. Based on logistic regression analysis, the minimum ROI-based ADC of 0.56 × 10(-3) mm(2)/s was a significant predictor for invasive disease (odds ratio = 0.02, 95% confidence interval [0.002, 0.207], P = .001). Regardless of the field strength (1.5 vs. 3.0 T) ADC values of high-grade and non-high-grade DCIS were not significantly different. CONCLUSIONS: Pure DCIS had the highest "ROI-based" ADC measured using 1.5 T or 3.0 T. The ADC was able to identify microinvasion or invasive cancer in biopsy-proven DCIS lesions but not to distinguish the DCIS grades.

Deficiency in Nrf2 transcription factor decreases adipose tissue mass and hepatic lipid accumulation in leptin-deficient mice.

OBJECTIVE: To evaluate whether Nrf2 deficiency impacts insulin resistance and lipid accumulation in liver and white adipose tissue. METHODS: Lep(ob/ob) mice (OB) with targeted Nrf2 deletion (OB-Nrf2KO) were generated. Pathogenesis of obesity and type 2 diabetes was measured in C57BL/6J, Nrf2KO, OB, and OB-Nrf2KO mice. Hepatic lipid content, lipid clearance, and very low-density lipoprotein (VLDL) secretion were determined between OB and OB-Nrf2KO mice. RESULTS: OB-Nrf2KO mice exhibited decreased white adipose tissue mass and decreased adipogenic and lipogenic gene expression compared with OB mice. Nrf2 deficiency prolonged hyperglycemia in response to glucose challenge, which was paralleled by reduced insulin-stimulated Akt phosphorylation. In OB mice, Nrf2 deficiency decreased hepatic lipid accumulation, decreased peroxisome proliferator-activated receptor γ expression and nicotinamide adenine dinucleotide phosphate (NADPH) content, and enhanced VLDL secretion. However, this observation was opposite in lean mice. Additionally, OB-Nrf2KO mice exhibited increased plasma triglyceride content, decreased HDL-cholesterol content, and enhanced apolipoprotein B expression, suggesting Nrf2 deficiency caused dyslipidemia in these mice. CONCLUSIONS: Nrf2 deficiency in Lep(ob/ob) mice reduced white adipose tissue mass and prevented hepatic lipid accumulation but induced insulin resistance and dyslipidemia. This study indicates a dual role of Nrf2 during metabolic dysregulation-increasing lipid accumulation in liver and white adipose tissue but preventing lipid accumulation in obese mice.

Fasting induces nuclear factor E2-related factor 2 and ATP-binding Cassette transporters via protein kinase A and Sirtuin-1 in mouse and human.

AIMS: The purpose of this study was to determine whether 3'-5'-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) and Sirtuin-1 (SIRT1) dependent mechanisms modulate ATP-binding Cassette (ABC) transport protein expression. ABC transport proteins (ABCC2-4) are essential for chemical elimination from hepatocytes and biliary excretion. Nuclear factor-E2 related-factor 2 (NRF2) is a transcription factor that mediates ABCC induction in response to chemical inducers and liver injury. However, a role for NRF2 in the regulation of transporter expression in nonchemical models of liver perturbation is largely undescribed. RESULTS: Here we show that fasting increased NRF2 target gene expression through NRF2- and SIRT1-dependent mechanisms. In intact mouse liver, fasting induces NRF2 target gene expression by at least 1.5 to 5-fold. In mouse and human hepatocytes, treatment with 8-Bromoadenosine-cAMP, a cAMP analogue, increased NRF2 target gene expression and antioxidant response element activity, which was decreased by the PKA inhibitor, H-89. Moreover, fasting induced NRF2 target gene expression was decreased in liver and hepatocytes of SIRT1 liver-specific null mice and NRF2-null mice. Lastly, NRF2 and SIRT1 were recruited to MAREs and Antioxidant Response Elements (AREs) in the human ABCC2 promoter. INNOVATION: Oxidative stress mediated NRF2 activation is well described, yet the influence of basic metabolic processes on NRF2 activation is just emerging. CONCLUSION: The current data point toward a novel role of nutrient status in regulation of NRF2 activity and the antioxidant response, and indicates that cAMP/PKA and SIRT1 are upstream regulators for fasting-induced activation of the NRF2-ARE pathway.

Effect of caloric restriction and AMPK activation on hepatic nuclear receptor, biotransformation enzyme, and transporter expression in lean and obese mice.

PURPOSE: Fatty liver alters liver transporter expression. Caloric restriction (CR), the recommended therapy to reverse fatty liver, increases Sirtuin1 deacetylase activity in liver. This study evaluated whether CR and CR mimetics reversed obesity-induced transporter expression in liver and hepatocytes. METHODS: mRNA and protein expression was determined in adult lean (lean) and leptin-deficient obese (OB) mice fed ad libitum or placed on 40% (kCal) reduced diet. Hepatocytes were isolated from lean and OB mice, treated with AMP Kinase activators, and gene expression was determined. RESULTS: CR decreased Oatp1a1, Oatp1b2, and Abcb11 mRNA expression in lean, but not OB mice. CR increased Abcc2 mRNA OB livers, whereas protein expression increased in both genotypes. CR increased Abcc3 protein expression increased in OB livers. CR did not alter Abcc1, 4 and 5 mRNA expression in lean mice but decreased expression in livers of OB mice. CR increased Abcc4 protein in lean, but not OB mice. CONCLUSIONS: CR restriction reversed the expression of some, but not all transporters in livers of OB mice. Overall, these data indicate a potential for CR to restore some hepatic transporter changes in OB mice, but suggest a functional leptin axis is needed for reversal of expression for some transporters.

Caloric restriction-mediated induction of lipid metabolism gene expression in liver is enhanced by Keap1-knockdown.

PURPOSE: CR increases fatty acid oxidation to decrease tissue lipid content. The Nuclear factor E2-related factor 2 (Nrf2)-Kelch like ECH associated Protein 1 (Keap1) pathway is an antioxidant gene regulatory pathway that has been previously investigated in weight gain. However, limited interaction of Nrf2/Keap1 and CR exists. The purpose of this study was to determine how Keap1 knockdown (Keap1-KD), which is known to increase Nrf2 activity, affects the CR response, such as weight loss, hepatic lipid decrease, and induction of fatty acid oxidation gene expression. METHODS: C57BL/6 and Keap1-KD mice were maintained on 40% CR or fed ad libitum for 6 weeks. Hepatic lipid content, lipid metabolic gene, and miRNA expression was quantified. RESULTS: CR lowered hepatic lipid content, and induced fatty acid oxidation gene expression to a greater degree in Keap1-KD compared to C57BL/6 mice. CR differentially altered miRNA 34a, 370, let-7b* in livers of Keap1-KD compared to C57BL/6 mice. CONCLUSIONS: CR induced induction of fatty acid oxidation gene expression was augmented with Keap1 knockdown, which was associated with differential expression of several miRNAs implicated in fatty acid oxidation and lipid accumulation.

Pilot study of quantitative analysis of background enhancement on breast MR images: association with menstrual cycle and mammographic breast density.

PURPOSE: To perform semiautomated quantitative analysis of the background enhancement (BE) in a cohort of patients with newly diagnosed breast cancer and to correlate it with mammographic breast density and menstrual cycle. MATERIALS AND METHODS: Informed consent was waived after the research ethics board approved this study. Results of 177 consecutive preoperative breast magnetic resonance (MR) examinations performed from February to December 2009 were reviewed; 147 female patients (median age, 48 years; range, 26-86 years) were included. Ordinal values of BE and breast density were described by two independent readers by using the Breast Imaging Reporting and Data System lexicon. The BE coefficient (BEC) was calculated thus: (SI2 · 100/SI1) - 100, where SI is signal intensity, SI2 is the SI enhancement measured in the largest anteroposterior dimension in the axial plane 1 minute after the contrast agent injection, and SI1is the SI before contrast agent injection. BEC was used for the quantitative analysis of BE. Menstrual cycle status was based on the last menstrual period. The Wilcoxon rank-sum or Kruskal-Wallis test was used to compare quantitative assessment groups. Cohen weighted κ was used to evaluate agreement. RESULTS: Of 147 patients, 68 (46%) were premenopausal and 79 (54%) were postmenopausal. The quantitative BEC was associated with the menstrual status (BEC in premenopausal women, 31.48 ± 20.68 [standard deviation]; BEC in postmenopausal women, 25.65 ± 16.74; P = .02). The percentage of overall BE was higher when the MR imaging was performed in women in the inadequate phase of the cycle (<35 days, not 7-14 days; mean BEC, 35.7) compared with women in the postmenopausal group (P = .001). Premenopausal women had significantly higher BEC when compared with postmenopausal women (P = .03). There was no significant difference in the percentage of BE between breast density groups. CONCLUSION: Premenopausal women with breast cancer, and specifically women in the inadequate phase of the cycle, presented with higher quantitative BE than postmenopausal women. No association was found between BE and breast density.

Incidence of breast cancer in patients with pure flat epithelial atypia diagnosed at core-needle biopsy of the breast.

PURPOSE: to determine the frequency of malignancy in subsequent breast excisions following core-needle biopsy (CNB) diagnosis of pure flat epithelial atypia (pFEA) and to evaluate the imaging features of the associated tumors. MATERIALS AND METHODS: Retrospective review of 8,996 image-guided CNB (2002-2010) identified 115 cases of FEA not associated with other atypia. Patients with history of breast cancer or radiation therapy were excluded. One hundred four cases (women) with pFEA (mean age 51 years, range 29-77 years) were reviewed. Stereotactic CNB was performed in 79 (76%) cases and ultrasound (US)-guided CNB in 25 (24%) cases. In 99 cases 14G needles were used, and 10G vacuum-assisted devices were used in 5 cases. Ninety-four patients had subsequent excision. Ten patients declined excision, and imaging follow-up (mean of 36 months) is available. The upgrade rate of pFEA was defined as the number of patients diagnosed with invasive carcinoma (IC) or carcinoma in situ (CIS) divided by the total number of patients. RESULTS: 10 of 104 (9.6%) patients were diagnosed with cancer: 9 presented as calcifications (89% fine pleomorphic and amorphous) and 1 case as a mammographically occult mass. The size of calcifications was not statistically significant (P=0.358). Five cases had ductal carcinoma in situ (DCIS) and five cases had IC (ductal and lobular) presenting as amorphous and pleomorphic calcifications. CONCLUSIONS: The upgrade rate of pFEA in our series was 9.6%. The presence of 4.8% of invasive cancers is substantial and warrants continuing management with surgical excision in all cases.

Enhanced Nrf2 activity worsens insulin resistance, impairs lipid accumulation in adipose tissue, and increases hepatic steatosis in leptin-deficient mice.

The study herein determined the role of nuclear factor erythoid 2-related factor 2 (Nrf2) in the pathogenesis of hepatic steatosis, insulin resistance, obesity, and type 2 diabetes. Lep(ob/ob)-Keap1-knockdown (KD) mice, which have increased Nrf2 activity, were generated. Markers of obesity and type 2 diabetes were measured in C57Bl/6J, Keap1-KD, Lep(ob/ob), and Lep(ob/ob)-Keap1-KD mice. Lep(ob/ob)-Keap1-KD mice exhibited less lipid accumulation, smaller adipocytes, decreased food intake, and reduced lipogenic gene expression. Enhanced Nrf2 activity impaired insulin signaling, prolonged hyperglycemia in response to glucose challenge, and induced insulin resistance in Lep(ob/ob) background. Nrf2 augmented hepatic steatosis and increased lipid deposition in liver. Next, C57Bl/6J and Keap1-KD mice were fed a high-fat diet (HFD) to determine whether Keap1 and Nrf2 impact HFD-induced obesity. HFD-induced obesity and lipid accumulation in white adipose tissue was decreased in Keap1-KD mice. Nrf2 activation via Keap1-KD or sulforaphane suppressed hormone-induced differentiation and decreased peroxisome proliferator-activated receptor-γ, CCAAT/enhancer-binding protein α, and fatty acid-binding protein 4 expression in mouse embryonic fibroblasts. Constitutive Nrf2 activation inhibited lipid accumulation in white adipose tissue, suppressed adipogenesis, induced insulin resistance and glucose intolerance, and increased hepatic steatosis in Lep(ob/ob) mice.

UDP-glucuronosyltransferase expression in mouse liver is increased in obesity- and fasting-induced steatosis.

UDP-glucuronosyltransferases (Ugt) catalyze phase II conjugation reactions with glucuronic acid, which enhances chemical polarity and the elimination from the body. Few studies have addressed whether Ugt expression and activity are affected by liver disease, such as steatosis. The purpose of this study was to determine whether steatosis induced by obesity or fasting could affect liver Ugt mRNA expression and activity. Male C57BL/6J and Lep(ob/ob) (ob/ob) mice were fed ad libitum or food was withheld for 24 h. In steatotic livers of ob/ob mice, Ugt1a1, -1a6, -1a9, -2a3, -3a1, and -3a2 mRNA expression increased. Fasting, which also induced steatosis, increased hepatic Ugt1a1, -1a6, -1a7, -1a9, -2b1, -2b5, -2a3, -3a1, and -3a2 mRNA expression in mouse liver. Likewise, acetaminophen glucuronidation increased by 47% in hepatic microsomes from ob/ob mice compared with that in C57BL/6J mice, but not after fasting. In both steatosis models, Ugt induction was accompanied by increased aryl hydrocarbon receptor, constitutive androstane receptor (CAR), peroxisome proliferator-activated receptor (PPAR)-α, pregnane X receptor, nuclear factor (erythroid-derived 2)-like 2 (Nrf2), and peroxisome proliferator-activated receptor-γ coactivator-1α mRNA expression. In addition, fasting increased CAR, PPAR, and Nrf2 binding activity. The work points to hepatic triglyceride concentrations corresponding with nuclear receptor and Ugt expression. The findings indicate that steatosis significantly alters hepatic Ugt expression and activity, which could have a significant impact on determining circulating hormone levels, drug efficacy, and environmental chemical clearance.

Accuracy of unenhanced MR imaging in the detection of axillary lymph node metastasis: study of reproducibility and reliability.

PURPOSE: To investigate the accuracy, reproducibility, and reliability of unenhanced magnetic resonance (MR) imaging techniques for detecting metastatic axillary lymph nodes in patients with newly diagnosed breast carcinoma. MATERIALS AND METHODS: Institutional review board approval and informed consent were obtained. Seventy-four consecutive women with invasive breast carcinoma were recruited to undergo preoperative breast MR imaging. Thirteen patients were excluded, two because they were undergoing preoperative chemotherapy and 11 because of the presence of movement or susceptibility artifacts on images. Thus, 61 patients (mean age, 53 years; range, 33-78 years) were included in this study. Axial T1-weighted MR images without fat saturation and diffusion-weighted (DW) MR images were analyzed by two experienced radiologists, who were blinded to the histopathologic findings. Visual and quantitative analyses of unenhanced MR images were performed. Sensitivity, specificity, and accuracy were calculated. To assess the intraobserver agreement, a second reading was performed. Statistical analysis was conducted on a patient-by-affected side basis. RESULTS: The sensitivity, specificity, and accuracy were 88%, 82%, and 85%, respectively, for axial T1-weighted MR imaging and 84%, 77%, and 80% for DW imaging. Apparent diffusion coefficients (ADCs) were significantly lower in the malignant group (P<.05 for all four readings), with the average of the four readings ranging from 0.333×10(-3) mm2/sec to 2.843×10(-3) mm2/sec. The mean Lin coefficient comparing the mean ADC reading for each observer was 0.959 (95% confidence interval: 0.935, 0.975), suggesting very high interobserver agreement between the two observers in terms of reproducibility of ADCs. The Bland-Altman plot showed good inter- and intraobserver agreement. CONCLUSION: Unenhanced MR imaging techniques showed high accuracy in the preoperative evaluation of axillary status in patients with invasive breast cancer. Results indicate reliable and reproducible assessment with DW imaging, but it is unlikely to be useful in clinical practice.