Mineralocorticoid receptor modulation by dietary sodium influences NAFLD development in mice.

INTRODUCTION AND OBJECTIVES: Nonalcoholic-fatty-liver disease (NAFLD) is considered the hepatic manifestation of metabolic syndrome (MetS). Mineralocorticoid receptor (MR) activation is associated with increased risk of MetS but few studies have assessed the role of liver MR on NAFLD. We aimed to evaluate the effect of MR modulation by sodium intake in liver injury in experimental models of NAFLD. MATERIALS AND METHODS: C57BL/6J mice were fed either a high-fat-diet (HFD) or a choline/methionine deficient (MCD) diet with different sodium concentrations. Hepatic concentration of lipid species, serum aldosterone levels, expression of MR, proinflammatory and profibrotic markers and liver histology were assessed. RESULTS: Mice fed with High-Na+/HFD showed a lower MR expression in liver (p = 0.01) and less steatosis on histology (p = 0.04). Consistently, animals from this group exhibited lower levels of serum aldosterone (p = 0.028) and lower hepatic triglyceride content (p = 0.008). This associated to a reduced expression of lipogenic genes, significant changes in lipid subspecies, lower HOMA-IR (p < 0.05), and lower expression of pro-inflammatory and profibrotic markers compared to those mice fed a Low-Na+/HFD. Additionally, mice fed a High-Na+/HFD showed higher expression of salt-inducible kinase (SIK)-1 and lower expression of serum-and-glucocorticoid-inducible kinase (SGK)-1. Similar results were observed with the MCD diet model. CONCLUSION: We identified in two experimental models of NAFLD that High-Na+ diet content is associated to lower serum aldosterone levels and hepatic MR downregulation, associated to decreased steatosis and reduced de novo hepatic lipogenesis, proinflammatory and profibrotic markers. Decreased activation of hepatic MR seems to generate beneficial downstream inhibition of lipogenesis in experimental NAFLD.

Somatotropic Axis Dysfunction in Non-Alcoholic Fatty Liver Disease: Beneficial Hepatic and Systemic Effects of Hormone Supplementation.

BACKGROUND: Somatotropic axis dysfunction associated with non-alcoholic fatty liver disease (NAFLD) has potential multisystemic detrimental effects. Here, we analysed the effects of growth hormone (GH) and insulin-like growth factor-1 (IGF-1) supplementation on liver histology, adipokine profile and muscle function in an NAFLD model. METHODS: C57BL/6 mice were fed with a high fat diet (HFD) for 12 weeks and were separated into three groups treated for 4 weeks with: (1) High fat diet (HFD) (n = 10); (2) HFD + GH 9 μg/g/d (n = 10); (3) HFD + IGF-1 0.02 µg/g/d (n = 9). A control group fed a chow diet was included (n = 6). Liver histology, liver triglycerides content, serum alanine aminotransferase (ALT) activity, adiponectin and leptin serum levels, in vivo muscle strength, tetanic force and muscle fibre cross-sectional area (CSA) were measured. RESULTS: HFD + GH and HFD + IGF-1 groups showed significantly lower ALT activity compared to HFD (p < 0.01). Liver triglyceride content in HFD + GH was decreased compared to HFD (p < 0.01). Histologic steatosis score was increased in HFD and HFD + GH group (p < 0.01), whereas HFD + IGF-1 presented no difference compared to the chow group (p = 0.3). HFD + GH group presented lower serum leptin and adiponectin levels compared to HFD. GH and IGF-1 supplementation therapy reverted HFD-induced reduction in muscle strength and CSA (sarcopenia). CONCLUSIONS: GH and IGF-1 supplementation induced significant improvement in liver steatosis, aminotransferases and sarcopenia in a diet-induced NAFLD model.

[Undergraduate student's perception of clinical simulation workshops: assessment of an instrument]. / Percepción de estudiantes de pregrado de Medicina de talleres de simulación de procedimientos médico-quirúrgicos.

BACKGROUND: Simulation is a useful training tool for undergraduate medical students. A valid instrument is needed to assess students' perception of simulation workshops. AIM: To adapt and validate an instrument to assess the undergraduate medical student's perception of simulation workshops of clinical procedures. MATERIAL AND METHODS: Delphi Methodology was used to adapt the instrument. Exploratory and confirmatory analyses were performed to determine the construct validity and Cronbach's Alpha (0 to 1) for internal consistency of the instrument. RESULTS: A Delphi panel of 10 experts adapted a seven-item questionnaire (Likert scale 1-5; ranging from 7 to 35) and four open-questions. After 3-delphi-rounds, the instrument was administered to 210 students in six simulation training programs (Paracentesis, Cardiopulmonary Resuscitation, Airway management, Sutures, Thoracentesis and Nursing Procedures). The instrument was considered unidimensional in the factorial analysis. The overall median (Q1-Q3) score was 34 ranging from 32 to 35 and the Cronbach Alpha coefficient was 0.72, indicating a good reliability. CONCLUSIONS: The perception questionnaire is a useful and reliable instrument to assess students' perceptions of clinical simulations.

Serum cytokeratin-18 fragment levels as noninvasive marker of nonalcoholic steatohepatitis in the chilean population. / Fragmento sérico de citoqueratina-18 como marcador no invasivo de esteatohepatitis no alcohólica en población chilena.

Nonalcoholic steatohepatitis (NASH) is the most aggressive form of nonalcoholic fatty liver disease (NAFLD) and involves the risk of progression to more advanced stages of liver disease. Non-invasive methods are needed to identify patients with NASH. OBJECTIVE: To evaluate the diagnostic performance of the determination of serum levels of cytokeratin-18 (CK-18) as a non-invasive marker of NASH in the Chilean population. METHODS: Serum CK-18 levels were determined in a group of 41 patients with biopsy-proven NAFLD. NASH diagnosis was based on Brunt's criteria (histological parameters and ballooning), and the NAFLD activity score (NAS) and the presence of fibrosis were determined. The correlation between the NAFLD activity score (NAS) and CK-18 was evaluated with Spearman's rank correlation coefficient. A ROC curve was produced to assess the diagnostic value of CK-18 for NASH. The NAFLD fibrosis score (NFS) (to predict fibrosis and NASH) was compared to CK-18 with simple linear regression. Data were expressed in median [25th-75th percentile] and evaluated with the Wilcoxon rank test. RESULTS: The mean age of the study group (23% male) was 50.4±11.1 years. 34.2% were diagnosed with NASH (NAS≥5). CK-18 levels were significantly higher in patients with NASH versus those without NASH (183.6 IU/l [97.4 to 734.4] vs. 117.2 IU/l [83.8 to 954.8], p= 0.016). CK-18 levels were a good predictor of NASH on biopsy with an area under the curve (AUC) of 0.732 (95% CI, 0.572 to 0.897). A CK-18 cut-off of 130.5 IU/l had a sensitivity of 92.9%, specificity of 63%, positive predictive value of 56.5% and negative predictive value of 94.4%, and was able to correctly classify 73.2% of patients with NASH. NFS identified advanced liver fibrosis (AUC 0.739, 95% CI, 0.56-0.91), but was of limited value to identify NASH (AUC 0.413, 95% CI, 0.21-0.61). CONCLUSION: CK-18 is a good non-invasive marker for NASH. Although NFS was found to be an accurate marker of advanced liver fibrosis, it was not of value to identify NASH. In patients with NAFLD, CK-18 and NFS could be useful in predicting NASH and liver fibrosis, respectively.

Diet-Induced Nonalcoholic Fatty Liver Disease Is Associated with Sarcopenia and Decreased Serum Insulin-Like Growth Factor-1.

BACKGROUND: Decreased muscle mass or sarcopenia has been associated with nonalcoholic fatty liver disease (NAFLD). However, the functional consequences of this association and its pathogenesis remain ill-defined. AIMS: To evaluate muscle mass and function in a diet-induced NAFLD mouse model and explore its association with changes in serum insulin-like growth factor-1 (IGF-1). METHODS: Weight gain, visceral fat, serum biochemical parameters, liver histology, and hepatic triglyceride content (HTC) were assessed in C57/Bl6 mice fed a westernized diet during 16 weeks. In addition, we determined muscle fiber size and strength of limb skeletal muscle, myosin heavy chain (MHC) protein levels, and IGF-1 serum levels. RESULTS: Westernized diet feeding was associated with weight gain, increased visceral fat mass (epididymal pad weight: 0.76 g ± 0.13 vs. 0.33 ± 0.27 g; p = 0.0023), hepatic steatosis (HTC: 118.2 ± 6.88 mg/g liver vs. 43.26 ± 5.63 mg/g<, p < 0.05), and necroinflammation (histological scores: 1.29 ± 0.42 vs. 4.00 ± 0.53<, p < 0.05). Also, mice fed the experimental diet had an increased proportion of low-diameter muscle fibers (0-30 µm) and a decreased proportion of high-diameter muscle fibers (60-90 µm), which correlated with decreased MHC protein levels, consistent with significant muscle atrophy. Functional studies showed that mice fed a westernized diet had reduced muscle strength and lower serum levels of IGF-1 (284.2 ± 20.04 pg/ml) compared with chow-fed mice (366.0 ± 12.42 pg/ml, p < 0.05). CONCLUSION: Experimental NAFLD is associated with sarcopenia, decreased muscle strength, and reduced IGF-1 serum levels. IGF-1 reduction may be involved in pathogenesis of NAFLD-associated sarcopenia.

Beneficial effects of mineralocorticoid receptor blockade in experimental non-alcoholic steatohepatitis.

BACKGROUND: Therapeutic options to treat Non-alcoholic steatohepatitis (NASH) are limited. Mineralocorticoid receptor (MR) activation could play a role in hepatic fibrogenesis and its modulation could be beneficial for NASH. AIM: To investigate whether eplerenone, a specific MR antagonist, ameliorates liver damage in experimental NASH. METHODS: C57bl6 mice were fed a choline-deficient and amino acid-defined (CDAA) diet for 22 weeks with or without eplerenone supplementation. Serum levels of aminotransferases and aldosterone were measured and hepatic steatosis, inflammation and fibrosis scored histologically. Hepatic triglyceride content (HTC) and hepatic mRNA levels of pro-inflammatory pro-fibrotic, oxidative stress-associated genes and of MR were also assessed. RESULTS: CDAA diet effectively induced fibrotic NASH, and increased the hepatic expression of pro-inflammatory, pro-fibrotic and oxidative stress-associated genes. Hepatic MR mRNA levels significantly correlated with the expression of pro-inflammatory and pro-fibrotic genes and were significantly increased in hepatic stellate cells obtained from CDAA-fed animals. Eplerenone administration was associated to a reduction in histological steatosis and attenuation of liver fibrosis development, which was associated to a significant decrease in the expression of collagen-α1, collagen type III, alpha 1 and Matrix metalloproteinase-2. CONCLUSION: The expression of MR correlates with inflammation and fibrosis development in experimental NASH. Specific MR blockade with eplerenone has hepatic anti-steatotic and anti-fibrotic effects. These data identify eplerenone as a potential novel therapy for NASH. Considering its safety and FDA-approved status, human studies are warranted.

[Introduction of virtual patients to clinical case portfolios for undergraduate medical students]. / Incorporación de paciente virtual en portafolio de estudiantes de medicina de pregrado.

BACKGROUND: In 2007, a Clinical-Case-Portfolio (CCP) was introduced as a new assessment instrument for fourth grade undergraduate medical students. Since then, several changes have been implemented such as reduction on the number of clinical cases, peer review and the introduction of virtual patient to the portfolio. AIM: To describe the virtual patient model incorporated to the CCP and assess the perception of this change and its effects on the performance of undergraduate students. MATERIAL AND METHODS: Virtual patients were implemented based on prototype clinical cases with specific syndromes. Students’ perceptions about CCP before and after the introduction of virtual patients were evaluated using a validated questionnaire that was answered voluntarily and anonymously. RESULTS: Overall perception of CCP significantly improved after the incorporation of virtual patients (97.1 ± 24.9 and 111.3 ± 25.7 points; 57.8 and 66.2% respectively). The same improvements were observed for the domains “Student Learning”, “Organization and Evaluation”, “Teaching Methodology” and “Integration”. In both years, students obtained high grades in CCP evaluations. However CCP grades were not significantly correlated with integrated final grades. CONCLUSIONS: The incorporation of virtual patients improved undergraduate students’ perception of CCP.

[Development of MEDUC-PG14 survey to assess postgraduate teaching in medical specialties]. / Evaluación de docentes clínicos de Postgrado: desarrollo y propiedades psicométricas del instrumento MEDUC-PG14.

BACKGROUND: Feedback is one of the most important tools to improve teaching in medical education. AIM: To develop an instrument to assess the performance of clinical postgraduate teachers in medical specialties. MATERIAL AND METHODS: A qualitative methodology consisting in interviews and focus-groups followed by a quantitative methodology to generate consensus, was employed. After generating the instrument, psychometric tests were performed to assess the construct validity (factor analysis) and reliability (Cronbach’s alpha). RESULTS: Experts in medical education, teachers and residents of a medical school participated in interviews and focus groups. With this information, 26 categories (79 items) were proposed and reduced to 14 items (Likert scale 1-5) by an expert’s Delphi panel, generating the MEDUC-PG14 survey, which was answered by 123 residents from different programs of medical specialties. Construct validity was carried out. Factor analysis showed three domains: Teaching and evaluation, respectful behavior towards patients and health care team, and providing feedback. The global score was 4.46 ± 0.94 (89% of the maximum). One teachers’ strength, as evaluated by their residents was “respectful behavior” with 4.85 ± 0.42 (97% of the maximum). “Providing feedback” obtained 4.09 ± 1.0 points (81.8% of the maximum). MEDUC-PG14 survey had a Cronbach’s alpha coefficient of 0.947. CONCLUSIONS: MEDUC-PG14 survey is a useful and reliable guide for teacher evaluation in medical specialty programs. Also provides feedback to improve educational skills of postgraduate clinical teachers.

[Assessment for learning: experience in an undergraduate medical theoretical course]. / Evaluación para el aprendizaje: experiencia en un curso teórico de pregrado en medicina.

BACKGROUND: Assessment for learning is a paradigm that is taking shape in the field of medical education. This approach aims to embed the assessment process within the educational and learning process. AIM: To evaluate the impact of curricular changes, from a focus of assessment of learning to one of assessment for learning, in the perception of undergraduate students of medicine and their final grades obtained in a theoretical course (TCG). MATERIAL AND METHODS: In the year 2011 lectures were reduced and intermediate assessments followed by a feedback session were introduced. The activities of each program course, surveys about student perceptions of the course and the final grades of students (assessments with multiple choice questions) were compared between the periods prior and after curricular changes (2005-2010 and 2011-2013). RESULTS: As a consequence of curricular changes, time for lectures was reduced by 19.5%, time for summative assessments was increased by 8.5%, and feedback activity, occupying 7.3% of the course time was added. There were significant improvements in student is perceptions in all areas assessed by surveys, emphasizing feedback and assessments. The overall grade assigned to the course dictated after implementing the changes increased from 6.18 to 6.59 (p < 0.001, 1-7 scale). The grades of students also improved from an average of 5.78 to 6.43 (p < 0.001, 1-7 scale). CONCLUSIONS: Assessment for learning achieved the desired educational impact without increasing the assigned curricular time. Programmatic assessment is favorably perceived by students.

Effects of bile acid sequestration on hepatic steatosis in obese mice.

BACKGROUND. Bile acid sequestration (BAS) with resins has shown antidiabetic effects in both humans and animals. Since hepatic steatosis is commonly associated with type 2 diabetes mellitus and the effects of BAS on steatosis have not been explored in detail, we evaluated the effects of cholestyramine (CTM) administration on fatty liver development in the leptin-deficient obese mice. AIM. To study the effects of BAS on fatty liver development in obese (ob/ob) mice. MATERIAL AND METHODS. 4 week-old ob/ob mice (B6.V-Lepob/J, n = 4-6 per group) were fed with or without CTM (control group) during 8 weeks. Serum and biliary parameters, glucose tolerance test (GTT), hepatic triglyceride content, liver histology and hepatic gene expression of relevant genes related to bile secretion, lipid and glucose metabolism were assessed. RESULTS. Control 12-week-old mice exhibited marked obesity and hepatic steatosis. CTM administration expectedly determined a marked de-repression of 7-α-hydroxylase and decreased biliary bile acid secretion as well as improved GTT. CTM feeding showed no effects on hepatic triglyceride content or in the degree of steatosis on liver histology. CTM was associated with increased levels of serum alanine-aminotransferase. CONCLUSION. Although CTM administration positively affects glucose tolerance it does not prevent hepatic steatosis development in obese mice. Moreover, CTM feeding was associated to liver enzyme elevation in this model of NAFLD. Thus, the effects BAS on NAFLD need to be specifically addressed since this therapy might not be beneficial for this condition.

Development of ACLEEM questionnaire, an instrument measuring residents' educational environment in postgraduate ambulatory setting.

BACKGROUND: Students' perceptions of their educational environment (EE) have been studied in undergraduate and postgraduate curricula. Postgraduate EE has been measured in hospital settings. However, there are no instruments available to measure the EE in postgraduate ambulatory settings. AIM: The aim of this study was to develop the "ambulatory care learning education environment measure" (ACLEEM). METHODS: A mixed methodology was used including three stages: (1) Grounded theory (focus groups); (2) Delphi technique to identify consensus; and (3) Pilot study. RESULTS: Three quota samples of approximately 60 stakeholders were formed, one as focus groups and two as Delphi panels. Eight focus groups were carried out including 58 residents (Latin-American Spanish speakers). The results were analysed and 173 items were offered to a National Delphi panel (61 residents and teachers). They reduced in two rounds the number of important items to 54. The 54-item questionnaire was then piloted with 63 residents and refined to the final version of the ACLEEM with 50 items and three domains. CONCLUSIONS: The 50-item inventory is a valid instrument to measure the EE in postgraduate ambulatory setting in Chile. Large-scale administration of the ACLEEM questionnaire to evaluate its construct validity and reliability are the next steps to test the psychometric properties of the instrument.

[Serum levels of alanine aminotransferase in Chilean population: analysis of results of the national health survey 2009-2010]. / Niveles séricos de alanino-aminotransferasa en población Chilena: análisis de los resultados de la encuesta nacional de salud 2009-2010.

BACKGROUND: Determination of Alanine aminotransferase serum levels ([ALT]s) is a sensitive ana reliable test for liver diseases. AIM: To report the prevalence of abnormal [ALT]s in Chilean population and to identify associated variables. METHODS: We analyzed data from a random sub-sample of 2,794 adults surveyed during the second Chilean National Health Survey. Abnormal [ALT]s were defined by using three different cut-off values (COV), two fixed COV (COV1: > 30 IU/L in men and > 19 IU/L in women and COV2 pre-defined by the performing laboratory) and a COV adjusted by age, weight and sex (COV3 > 31 IU/L for women and > 44 IU/L and men > 42 IU/L and > 66 IU/L with a BMI > 23). Logistic regression analysis was performed to determine risk factors for elevated [ALT]s. RESULTS: Mean [ALT]s values were 30.14 I U/L in men and 22.03 IU/L in women. The observed prevalence of abnormal [ALT]s defined by different COV were 38%, 11.5%, and 8.1% for COV1, COV2 and COV3 respectively. Variables independently associated to abnormal [ALT]s in a multivariate analysis were the following: serum gamma-glutamyl-transpeptidase (OR: 1.055 [95% CI 1.033-1.078]) and body mass index (OR:1.13 [95% CI 1.09-1.17]). Variables inversely associated with abnormal [ALT]s (COV1) were mole gender (OR-.0.976 [95% CI 0.96-0.99) and HDL-cholesterol (OR:0979 [95% CI 0.96-0.99]). CONCLUSIONS: Independently of the COV used, Chilean population exhibits a high prevalence of abnormal [ALT]s which may reflect a significant burden of liver disease. Non-alcoholic fatty liver disease could be a major contributor to elevated [ALT]s considering the association of abnormal [ALT]s and metabolic variables.

[Identifying the main training needs of postgraduate medical program managers]. / Identificación de las necesidades de capacitación docente de los jefes de programa de especialización médica.

BACKGROUND: Training of postgraduate medical specialty program managers (PMSPM) is essential for the proper development of their programs. AIM: To identify the main training needs of PMSPM at a medical school. MATERIAL AND METHODS: A mixed-methodology approach was implemented including focus group/interviews and the administration of the Program Managers Training Needs Assessment Questionnaire (PROMANAQ) developed by an expert panel with 59 items (with two sections: relevance/performance-self-perception). Higher priority was assigned to items with high relevance and low performance. RESULTS: Forty five PMSPM completed the PROMANAQ (81.8% response rate). Both sections of PROMANAQ were highly reliable (Cronbach alpha of 0.95/0.97 for relevance/performance-self-perception, respectively). The items with higher priority value were evaluation of clinical educators, evaluation of teaching programs and accreditation of programs. Ten PMSPM were included in the focus group (18.2% of the universe). The findings of the qualitative component were concordant with the areas explored in the questionnaire. CONCLUSIONS: The PROMANAQ is valid and reliable to identify the training needs of PMSPM. The views of PMSPM must be taken into account for faculty development planning.

Overexpression of 11ß-hydroxysteroid dehydrogenase type 1 in visceral adipose tissue and portal hypercortisolism in non-alcoholic fatty liver disease.

BACKGROUND: The enzyme 11ß-hydroxysteroid-dehydrogenase type 1 (11ß-HSD1) catalyses the reactivation of intracellular cortisol. We explored the potential role of 11ß-HSD1 overexpression in visceral adipose tissue (VAT) in non-alcoholic fatty liver disease (NAFLD) assessing sequential changes of enzyme expression, in hepatic and adipose tissue, and the occurrence of portal hypercortisolism in obese mice. 11ß-HSD1 expression was also assessed in tissues from obese patients undergoing bariatric surgery. METHODS: Peripheral and portal corticosterone levels and liver histology were assessed in ob/ob mice at two time points (8-12 weeks of age). 11ß-HSD1 tissue expression was assessed in by RT-pcr in ob/ob mice and in 49 morbidly obese patients. RESULTS: Portal corticosterone serum levels were higher in obese mice with a 26% decrease between 8 and 12 weeks of age (controls: 78.3 ± 19.7 ng/ml, 8-week-old ob/ob: 167.5 ± 14.5 ng/ml and 12-week-old ob/ob: 124.3 ± 28 ng/ml, P < 0.05). No significant differences were found in peripheral corticosterone serum levels. Expression of 11ß-HSD1 was lower in the liver [-45% at 8 weeks and -35% at 12-weeks (P = 0.0001)] and highly overexpressed in VAT in obese mice, compared to controls (128-fold higher in 8-week-old ob/ob and 41-fold higher in 12-week-old ob/ob, P < 0.01). No significant differences were seen in the expression of 11ß-HSD1 in subcutaneous adipose tissue. In multivariate analysis, human 11ß-HSD1 expression in VAT (OR: 1.385 ± 1.010-1.910) was associated with NAFLD. CONCLUSION: Murine NAFLD is associated with portal hypercortisolism and11ß-HSD1 overexpression in VAT. In humans, 11ß-HSD1 VAT expression was associated with the presence of NAFLD. Thus, local corticosteroid production in VAT may contribute to NAFLD pathogenesis.

[Evaluation of the educational environment in medical specialty programs]. / Evaluación del ambiente educacional en programas de especialización médica.

BACKGROUND: The Postgraduate Hospital Education Environment Measure (PHEEM) questionnaire, is a valid and reliable instrument to measure the educational environment (EE) in postgraduate medical education. AIM: To evaluate the EE perceived by the residents of a postgraduate training program using the PHEEM. MATERIAL AND METHODS: The PHEEM was applied in 2010-2011 in 35 specialty programs. We calculated their individual results and compared means of both global and individual domain scores of the PHEEM, by gender, university of origin and nationality. Cronbach's alpha coefficients and D study (Generalizability theory) were performed for reliability. RESULTS: Three hundred eighteen residents were surveyed (75.7% of the total universe). The mean score of the PHEEM was 105.09 ± 22.46 (65.7% of the maximal score) which is considered a positive EE. The instrument is highly reliable (Cronbach's alpha = 0.934). The D study found that 15 subjects are required to obtain reliable results (G coefficient = 0.813). There were no significant differences between gender and university of origin. Foreigners evaluated better the EE than Chileans and racism was not perceived. The programs showed a safe physical environment and teachers with good clinical skills. The negative aspects perceived were a lack of information about working hours, insufficient academic counseling, and scanty time left for extracurricular activities. CONCLUSIONS: This questionnaire allowed us to identify positive aspects of the EE, and areas to be improved in the specialty programs. The PHEEM is a useful instrument to evaluate the EE in Spanish-speaking participants of medical specialty programs.

Plasminogen activator inhibitor type 1 serum levels and 4G/5G gene polymorphism in morbidly obese Hispanic patients with non-alcoholic fatty liver disease.

BACKGROUND: The plasminogen activator inhibitor type-1 (PAI-1) has been implicated in the regulation of fibrinolysis and extracellular matrix components. The single base pair guanine insertion/deletion polymorphism (4G/5G) within the promoter region of the PAI-1 gene influences PAI-1 synthesis and may modulate hepatic fibrogenesis. AIM: To evaluate the influence of PAI-1 serum levels and 4G/5G polymorphism on the risk of liver fibrosis associated to non-alcoholic fatty liver disease (NAFLD) in morbidly obese patients. MATERIAL AND METHODS: Case-control study of 50 obese patients undergoing bariatric surgery and 71 non-obese subjects matched by age and sex. Anthropometric and biochemical measurements were performed, including PAI-1 serum levels. Genomic DNA was obtained to assess the presence of 4G/5G polymorphism. RESULTS: BMI, insulinemia, triglycerides, HOMA-IR, hypertension and diabetes were significantly higher in obese patients compared to control subjects. PAI-1 serum levels observed in obese patients were significantly lower (10.63 ± 4.82) compared to controls (14.26 ± 11.4; p < 0.05). No differences were observed in the PAI-1 4G/5G promoter genotypes frequencies (p = 0.12). No differences were observed in PAI-1 plasma levels among obese patients with liver fibrosis (10.64 ± 4.35) compared to patients without liver fibrosis (10.61 ± 5.2; p = 0.985). PAI-1 4G/5G promoter genotypes frequencies were similar in patients with or without liver fibrosis associated to NASH (p = 0.6). CONCLUSIONS: Morbidly obese patients had significantly lower PAI-1 serum levels with similar PAI-1 4G/5G genotypes frequencies compared to non-obese subjects. The frequency of 4G/5G genotypes in Chilean Hispanic healthy subjects was similar to that described in other populations. No association was found between PAI-1 serum levels or 4G/5G genotype with liver fibrosis in obese patients.

Farnesoid X receptor critically determines the fibrotic response in mice but is expressed to a low extent in human hepatic stellate cells and periductal myofibroblasts.

The nuclear bile acid receptor, farnesoid X receptor (FXR), may play a pivotal role in liver fibrosis. We tested the impact of genetic FXR ablation in four different mouse models. Hepatic fibrosis was induced in wild-type and FXR knock-out mice (FXR(-/-)) by CCl(4) intoxication, 3,5-diethoxycarbonyl-1,4-dihydrocollidine feeding, common bile duct ligation, or Schistosoma mansoni (S.m.)-infection. In addition, we determined nuclear receptor expression levels (FXR, pregnane X receptor (PXR), vitamin D receptor, constitutive androstane receptor (CAR), small heterodimer partner (SHP)) in mouse hepatic stellate cells (HSCs), portal myofibroblasts (MFBs), and human HSCs. Cell type-specific FXR protein expression was determined by immunohistochemistry in five mouse models and prototypic human fibrotic liver diseases. Expression of nuclear receptors was much lower in mouse and human HSCs/MFBs compared with total liver expression with the exception of vitamin D receptor. FXR protein was undetectable in mouse and human HSCs and MFBs. FXR loss had no effect in CCl(4)-intoxicated and S.m.-infected mice, but significantly decreased liver fibrosis of the biliary type (common bile duct ligation, 3,5-diethoxycarbonyl-1,4-dihydrocollidine). These data suggest that FXR loss significantly reduces fibrosis of the biliary type, but has no impact on non-cholestatic liver fibrosis. Since there is no FXR expression in HSCs and MFBs in liver fibrosis, our data indicate that these cells may not represent direct therapeutic targets for FXR ligands.

Chemical hypoxia induces pro-inflammatory signals in fat-laden hepatocytes and contributes to cellular crosstalk with Kupffer cells through extracellular vesicles.

BACKGROUND: Obstructive sleep apnea syndrome (OSAS) is associated to intermittent hypoxia (IH) and is an aggravating factor of non-alcoholic fatty liver disease (NAFLD). We investigated the effects of hypoxia in both in vitro and in vivo models of NAFLD. METHODS: Primary rat hepatocytes treated with free fatty acids (FFA) were subjected to chemically induced hypoxia (CH) using the hypoxia-inducible factor-1 alpha (HIF-1α) stabilizer cobalt chloride (CoCl2). Triglyceride (TG) content, mitochondrial superoxide production, cell death rates, cytokine and inflammasome components gene expression and protein levels of cleaved caspase-1 were assessed. Also, Kupffer cells (KC) were treated with conditioned medium (CM) and extracellular vehicles (EVs) from hypoxic fat-laden hepatic cells. The choline deficient L-amino acid defined (CDAA)-feeding model used to assess the effects of IH on experimental NAFLD in vivo. RESULTS: Hypoxia induced HIF-1α in cells and animals. Hepatocytes exposed to FFA and CoCl2 exhibited increased TG content and higher cell death rates as well as increased mitochondrial superoxide production and mRNA levels of pro-inflammatory cytokines and of inflammasome-components interleukin-1ß, NLRP3 and ASC. Protein levels of cleaved caspase-1 increased in CH-exposed hepatocytes. CM and EVs from hypoxic fat-laden hepatic cells evoked a pro-inflammatory phenotype in KC. Livers from CDAA-fed mice exposed to IH exhibited increased mRNA levels of pro-inflammatory and inflammasome genes and increased levels of cleaved caspase-1. CONCLUSION: Hypoxia promotes inflammatory signals including inflammasome/caspase-1 activation in fat-laden hepatocytes and contributes to cellular crosstalk with KC by release of EVs. These mechanisms may underlie the aggravating effect of OSAS on NAFLD. [Abstract word count: 257].

Extracellular vesicles derived from fat-laden hepatocytes undergoing chemical hypoxia promote a pro-fibrotic phenotype in hepatic stellate cells.

BACKGROUND: The transition from steatosis to non-alcoholic steatohepatitis (NASH) is a key issue in non-alcoholic fatty liver disease (NAFLD). Observations in patients with obstructive sleep apnea syndrome (OSAS) suggest that hypoxia contributes to progression to NASH and liver fibrosis, and the release of extracellular vesicles (EVs) by injured hepatocytes has been implicated in NAFLD progression. AIM: To evaluate the effects of hypoxia on hepatic pro-fibrotic response and EV release in experimental NAFLD and to assess cellular crosstalk between hepatocytes and human hepatic stellate cells (LX-2). METHODS: HepG2 cells were treated with fatty acids and subjected to chemically induced hypoxia using the hypoxia-inducible factor 1 alpha (HIF-1α) stabilizer cobalt chloride (CoCl2). Lipid droplets, oxidative stress, apoptosis and pro-inflammatory and pro-fibrotic-associated genes were assessed. EVs were isolated by ultracentrifugation. LX-2 cells were treated with EVs from hepatocytes. The CDAA-fed mouse model was used to assess the effects of intermittent hypoxia (IH) in experimental NASH. RESULTS: Chemical hypoxia increased steatosis, oxidative stress, apoptosis and pro-inflammatory and pro-fibrotic gene expressions in fat-laden HepG2 cells. Chemical hypoxia also increased the release of EVs from HepG2 cells. Treatment of LX2 cells with EVs from fat-laden HepG2 cells undergoing chemical hypoxia increased expression pro-fibrotic markers. CDAA-fed animals exposed to IH exhibited increased portal inflammation and fibrosis that correlated with an increase in circulating EVs. CONCLUSION: Chemical hypoxia promotes hepatocellular damage and pro-inflammatory and pro-fibrotic signaling in steatotic hepatocytes both in vitro and in vivo. EVs from fat-laden hepatocytes undergoing chemical hypoxia evoke pro-fibrotic responses in LX-2 cells.

Mild hypothermia does not affect liver regeneration after partial hepatectomy in mice.

BACKGROUND: The use of mild hypothermia has been suggested to be therapeutically useful in treating acute liver failure. It is not known if hypothermia influences liver regeneration. AIM: To assess the effect of hypothermia on liver regeneration in mice. METHODS: After partial (70%) hepatectomy (PHx), C57BL6/J mice were randomly assigned to either a hypothermic group or a normothermic group. Controlled mild hypothermia was maintained for up to 3 h after surgery. In addition, assessment of liver mass restitution was examined by studying the induction of key cell cycle proteins (cyclin A, D1 and E) and hepatocyte proliferation [assessment of proliferating cell nuclear antigen (PCNA) protein expression] by Western blotting and DNA synthesis by measuring 5-bromo-2-deoxyuridine (BrdU) incorporation by immunohistochemical techniques 45 h after PHx. RESULTS: Partial hepatectomy induced a vigorous proliferative response in the remnant livers of both groups of mice (normothermic and hypothermic groups), as evidenced by the induction of key cyclins, PCNA and incorporation of BrdU after PHx. The liver/body weight ratio and both cyclin and PCNA protein expression as well as BrdU incorporation did not differ between the regenerating livers of hypothermic and normothermic groups. CONCLUSION: Mild hypothermia does not influence liver regeneration in mice.