Alteração do tecido adiposo e fígado em modelo experimental de síndrome metabólica: ação de agonista PPAR-gama e bloqueador de receptor AT1 da angiotensina 2

Este trabalho teve como objetivo investigar os efeitos da telmisartana (agonista PPAR-gama parcial), losartana (puro bloqueador do receptor AT1 da angiotensina II) e rosiglitazona (agonista PPAR-gama) em modelo experimental de síndrome metabólica. Os alvos do estudo foram a pressão arterial, metabolismo de carboidratos, resistência insulínica, inflamação, tecido adiposo e fígado. Camundongos C57BL/6 (a partir de 3 meses de idade) foram alimentados com dieta padrão (SC, n = 10) ou dieta hiperlipídica rica em sal (HFHS, n = 40) por 12 semanas. Após esse tempo, os animais do grupo HFHS foram subdivididos em 4 grupos (n = 10): HFHS (sem tratamento), ROSI (HFHS tratado com rosiglitazona), TELM (HFHS tratado com telmisartana) e LOS (HFHS tratado com losartana) por 5 semanas. O grupo HFHS apresentou um significante ganho de peso e aumento da pressão arterial sistólica, hiperinsulinemia com resistência insulínica, hiperleptinemia, hipertrofia de adipócitos bem como um quadro de esteatose hepática e níveis aumentados da citocina inflamatória interleucina-6 (IL-6). Os animais tratados com telmisartana chegou ao final do experimento com massa corporal similar ao grupo SC, com reversão do quadro de resistência insulínica, com pressão arterial normal, adipócitos de tamanho normal e sem apresentar esteatose hepática. Além disso, o tratamento com telmisartana aumentou a expressão de PPARγ e adiponectina no tecido adiposo epididimal. A expressão da proteína desacopladora-1 (UCP-1) no tecido adiposo branco (TAB) também foi aumentada. O tratamento com losartana diminuiu a pressão arterial para valores normais, porém com menores efeitos nos parâmetros metabólicos dos animais. O presente modelo experimental de ganho de peso e hipertensão induzidos por dieta mimetiza a síndrome metabólica humana. Neste modelo, a telmisartana aumentou a expressão de UCP-1 no TAB, preveniu o ganho de peso e melhorou a sensibilidade à insulina e a esteatose hepática dos camundongos C57BL/6, provavelmente devido à ativação PPAR-gama.

Differences in Abdominal and Liver Fat Accumulation Between Obesity-Matched Adults With and Without Type 2 Diabetes

Despite attempts to identify the mechanisms by which obesity leads to the development of Type 2 Diabetes (T2D), it remains unclear why some but not all adults with obesity develop T2D. Given the established associations between visceral adipose tissue (VAT) and liver fat with insulin resistance, we hypothesized that compared to age and obesity matched adults who were non-diabetic (NT2D), adults with T2D would have greater amounts of VAT and liver fat. The International Study of Prediction of Intra-Abdominal Adiposity and Its Relationship with Cardiometabolic Risk/Intra-Abdominal Adiposity (INSPIRE ME IAA) aims to study the associations between VAT and liver fat and risk of developing T2D and cardiovascular disease. Four thousand, five hundred and four participants were initially recruited; from this, 2383 White and Asian adults were selected for this ancillary analysis. The NT2D and T2D groups were matched for age, body mass index (BMI) and waist circumference (WC). The T2D and NT2D groups were also compared to participants with either impaired fasting glucose (IFG) or impaired glucose tolerance (IGT; IFG/IGT)). Abdominal adipose tissue was measured by computed tomography; liver fat was estimated using computed tomography-derived mean attenuation. Secondary analysis determined whether differences existed between NT2D and T2D groups in VAT and liver fat accumulation within selected BMI categories for Whites and Asians. We report across sex and race, T2D and IFG/IGT groups had elevated VAT and liver fat compared to the NT2D group (p<0.05). VAT was not different between IFG/IGT and T2D groups (p>0.05), however liver fat was greater in the T2D group compared to the IFG/IGT group in both Whites and Asians (p<0.05). Within each BMI category, the T2D group had elevated VAT and liver fat compared to the age and anthropometrically matched NT2D group in both Whites and Asians (p<0.05). With few exceptions, abdominal subcutaneous adipose tissue was not different in the T2D or IFG/IGT groups compared to the NT2D group independent of sex and race. Compared to age and obesity-matched adults who are NT2D, we observe that White and Asian adults with T2D, and those with IFG/IGT, present with greater levels of both VAT and liver fat.

NEW TARGET GENES FOR TUMOR SUPPRESSORS p53 AND p73 IN REGENERATING LIVER

The p53-family of proteins regulates expression of target genes during tissue development and differentiation. Within the p53-family, p53 and p73 have hepatic-specific functions in development and tumor suppression. Despite a growing list of p53/p73 target genes, very few of these have been studied in vivo, and the knowledge regarding functions of p53 and p73 in normal tissues remains limited. p53+/-p73+/- mice develop hepatocellular carcinoma (HCC), whereas overexpression of p53 in human HCC leads to tumor regression. However, the mechanism of p53/p73 function in liver remains poorly characterized. Here, the model of mouse liver regeneration is used to identify new target genes for p53/p73 in normal quiescent vs. proliferating cells. In response to surgical removal of ~2/3 of liver mass (partial hepatectomy, PH), the remaining hepatocytes exit G0 of cell cycle and undergo proliferation to reestablish liver mass. The hypothesis tested in this work is that p53/p73 functions in cell cycle arrest, apoptosis and senescence are repressed during liver regeneration, and reactivated at the end of the regenerative response. Chromatin immunoprecipitation (ChIP), with a p73-antibody, was used to probe arrayed genomic sequences (ChIP-chip) and uncover 158 potential targets of p73-regulation in normal liver. Global microarray analysis of mRNA levels, at T=0-48h following PH, revealed sets of genes that change expression during regeneration. Eighteen p73-bound genes changed expression after PH. Four of these genes, Foxo3, Jak1, Pea15, and Tuba1 have p53 response elements (p53REs), identified in silico within the upstream regulatory region. Forkhead transcription factor Foxo3 is the most responsive gene among transcription factors with altered expression during regenerative, cellular proliferation. p53 and p73 bind a Foxo3 p53RE and maintain active expression in quiescent liver. During liver regeneration, binding of p53 and p73, recruitment of acetyltransferase p300, and an active chromatin structure of Foxo3 are disrupted, alongside loss of Foxo3 expression. These parameters of Foxo3 regulation are reestablished at completion of liver growth and regeneration, supporting a temporary suspension of p53 and p73 regulatory functions in normal cells during tissue regeneration.

GENETIC ANALYSIS OF THE HIPPO PATHWAY IN MOUSE LIVER

Cancer therapy and tumor treatment remain unsolved puzzles. Genetic screening for tumor suppressor genes in Drosophila revealed the Hippo-signaling pathway as a kinase cascade consisting of five core components. Disrupting the pathway by deleting the main component genes breaks the balance of cell proliferation and apoptosis and results in epithelial tissue tumorigenesis. The pathway is therefore believed to be a tumor suppressor pathway. However, a corresponding role in mammals is yet to be determined. Our lab began to investigate the tumor suppression function of the potent mammalian Hippo pathway by putting floxed alleles into the mouse genome flanking the functional-domain-expressing exons in each component (Mst1, Mst2, Sav1, Lats1 and Lats2). These mice were then crossed with different cre-mouse lines to generate conditional knockout mice. Results indicate a ubiquitous tumor suppression function of these components, predominantly in the liver. A further liver specific analysis of the deletion mutation of these components, as well as the Yap/Taz double deletion mutation, reveals essential roles of the Hippo pathway in regulating hepatic quiescence and embryonic liver development. One of the key cellular mechanisms for the Hippo pathway’s involvement in these liver biological events is likely its cell cycle regulation function. Our work will help to develop potential therapeutic approaches for liver cancer.

Microspheres for Liver Radiomicrospheres Therapy and Planning

Liver cancer accounts for nearly 10% of all cancers in the US. Intrahepatic Arterial Radiomicrosphere Therapy (RMT), also known as Selective Internal Radiation Treatment (SIRT), is one of the evolving treatment modalities. Successful patient clinical outcomes require suitable treatment planning followed by delivery of the microspheres for therapy. The production and in vitro evaluation of various polymers (PGCD, CHS and CHSg) microspheres for a RMT and RMT planning are described. Microparticles with a 30±10 µm size distribution were prepared by emulsion method. The in vitro half-life of the particles was determined in PBS buffer and porcine plasma and their potential application (treatment or treatment planning) established. Further, the fast degrading microspheres (≤ 48 hours in vitro half-life) were labeled with 68Ga and/or 99mTc as they are suitable for the imaging component of treatment planning, which is the primary emphasis of this dissertation. Labeling kinetics demonstrated that 68Ga-PGCD, 68Ga-CHSg and 68Ga-NOTA-CHSg can be labeled with more than 95% yield in 15 minutes; 99mTc-PGCD and 99mTc-CHSg can also be labeled with high yield within 15-30 minutes. In vitro stability after four hours was more than 90% in saline and PBS buffer for all of them. Experiments in reconstituted hemoglobin lysate were also performed. Two successful imaging (RMT planning) agents were found: 99mTc-CHSg and 68Ga-NOTA-CHSg. For the 99mTc-PGCD a successful perfusion image was obtained after 10 minutes, however the in vivo degradation was very fast (half-life), releasing the 99mTc from the lungs. Slow degrading CHS microparticles (> 21 days half-life) were modified with p-SCN-b-DOTA and labeled with 90Y for production of 90Y-DOTA-CHS. Radiochemical purity was evaluated in vitro and in vivo showing more than 90% stability after 72 and 24 hours respectively. All agents were compared to their respective gold standards (99mTc-MAA for 68Ga-NOTA-CHSg and 99mTc-CHSg; 90Y-SirTEX for 90Y-DOTA-CHS) showing superior in vivo stability. RMT and RMT planning agents (Therapy, PET and SPECT imaging) were designed and successfully evaluated in vitro and in vivo.

Non-alcoholic fatty liver disease : can ultrasound assist in early diagnosis of prediabetes and delay progression to type2 diabetes mellitus

Non Alcoholic Fatty Liver Disease (NAFLD) is a condition that is frequently seen but seldom investigated. Until recently, NAFLD was considered benign, self-limiting and unworthy of further investigation. This opinion is based on retrospective studies with relatively small numbers and scant follow-up of histology data. (1) The prevalence for adults, in the USA is, 30%, and NAFLD is recognized as a common and increasing form of liver disease in the paediatric population (1). Australian data, from New South Wales, suggests the prevalence of NAFLD in “healthy” 15 year olds as being 10%.(2) Non-alcoholic fatty liver disease is a condition where fat progressively invades the liver parenchyma. The degree of infiltration ranges from simple steatosis (fat only) to steatohepatitis (fat and inflammation) steatohepatitis plus fibrosis (fat, inflammation and fibrosis) to cirrhosis (replacement of liver texture by scarred, fibrotic and non functioning tissue).Non-alcoholic fatty liver is diagnosed by exclusion rather than inclusion. None of the currently available diagnostic techniques -liver biopsy, liver function tests (LFT) or Imaging; ultrasound, Computerised tomography (CT) or Magnetic Resonance Imaging (MRI) are specific for non-alcoholic fatty liver. An association exists between NAFLD, Non Alcoholic Steatosis Hepatitis (NASH) and irreversible liver damage, cirrhosis and hepatoma. However, a more pervasive aspect of NAFLD is the association with Metabolic Syndrome. This Syndrome is categorised by increased insulin resistance (IR) and NAFLD is thought to be the hepatic representation. Those with NAFLD have an increased risk of death (3) and it is an independent predictor of atherosclerosis and cardiovascular disease (1). Liver biopsy is considered the gold standard for diagnosis, (4), and grading and staging, of non-alcoholic fatty liver disease. Fatty-liver is diagnosed when there is macrovesicular steatosis with displacement of the nucleus to the edge of the cell and at least 5% of the hepatocytes are seen to contain fat (4).Steatosis represents fat accumulation in liver tissue without inflammation. However, it is only called non-alcoholic fatty liver disease when alcohol - >20gms-30gms per day (5), has been excluded from the diet. Both non-alcoholic and alcoholic fatty liver are identical on histology. (4).LFT’s are indicative, not diagnostic. They indicate that a condition may be present but they are unable to diagnosis what the condition is. When a patient presents with raised fasting blood glucose, low HDL (high density lipoprotein), and elevated fasting triacylglycerols they are likely to have NAFLD. (6) Of the imaging techniques MRI is the least variable and the most reproducible. With CT scanning liver fat content can be semi quantitatively estimated. With increasing hepatic steatosis, liver attenuation values decrease by 1.6 Hounsfield units for every milligram of triglyceride deposited per gram of liver tissue (7). Ultrasound permits early detection of fatty liver, often in the preclinical stages before symptoms are present and serum alterations occur. Earlier, accurate reporting of this condition will allow appropriate intervention resulting in better patient health outcomes. References 1. Chalasami N. Does fat alone cause significant liver disease: It remains unclear whether simple steatosis is truly benign. American Gastroenterological Association Perspectives, February/March 2008 www.gastro.org/wmspage.cfm?parm1=5097 Viewed 20th October, 2008 2. Booth, M. George, J.Denney-Wilson, E: The population prevalence of adverse concentrations with adiposity of liver tests among Australian adolescents. Journal of Paediatrics and Child Health.2008 November 3. Catalano, D, Trovato, GM, Martines, GF, Randazzo, M, Tonzuso, A. Bright liver, body composition and insulin resistance changes with nutritional intervention: a follow-up study .Liver Int.2008; February 1280-9 4. Choudhury, J, Sanysl, A. Clinical aspects of Fatty Liver Disease. Semin in Liver Dis. 2004:24 (4):349-62 5. Dionysus Study Group. Drinking factors as cofactors of risk for alcohol induced liver change. Gut. 1997; 41 845-50 6. Preiss, D, Sattar, N. Non-alcoholic fatty liver disease: an overview of prevalence, diagnosis, pathogenesis and treatment considerations. Clin Sci.2008; 115 141-50 7. American Gastroenterological Association. Technical review on nonalcoholic fatty liver disease. Gastroenterology.2002; 123: 1705-25

In vitro estimation of foetal liver volume using ultrasound, x-ray computed tomography and magnetic resonance imaging

Sixteen formalin-fixed foetal livers were scanned in vitro using a new system for estimating volume from a sequence of multiplanar 2D ultrasound images. Three different scan techniques were used (radial, parallel and slanted) and four volume estimation algorithms (ellipsoid, planimetry, tetrahedral and ray tracing). Actual liver volumes were measured by water displacement. Twelve of the sixteen livers also received x-ray computed tomography (CT) and magnetic resonance (MR) scans and the volumes were calculated using voxel counting and planimetry. The percentage accuracy (mean ± SD) was 5.3 ± 4.7%, −3.1 ± 9.6% and −0.03 ± 9.7% for ultrasound (radial scans, ray volumes), MR and CT (voxel counting) respectively. The new system may be useful for accurately estimating foetal liver volume in utero.

Whole-body substrate metabolism is associated with disease severity in patients with non-alcoholic fatty liver disease

Objectives In non-alcoholic fatty liver disease (NAFLD), hepatic steatosis is intricately linked with a number of metabolic alterations. We studied substrate utilisation in NAFLD during basal, insulin-stimulated and exercise conditions, and correlated these outcomes with disease severity. Methods 20 patients with NAFLD (mean±SD body mass index (BMI) 34.1±6.7 kg/m2) and 15 healthy controls (BMI 23.4±2.7 kg/m2) were assessed. Respiratory quotient (RQ), whole-body fat (Fatox) and carbohydrate (CHOox) oxidation rates were determined by indirect calorimetry in three conditions: basal (resting and fasted), insulin-stimulated (hyperinsulinaemic–euglycaemic clamp) and exercise (cycling at an intensity to elicit maximal Fatox). Severity of disease and steatosis were determined by liver histology, hepatic Fatox from plasma β-hydroxybutyrate concentrations, aerobic fitness expressed as , and visceral adipose tissue (VAT) measured by computed tomography. Results Within the overweight/obese NAFLD cohort, basal RQ correlated positively with steatosis (r=0.57, p=0.01) and was higher (indicating smaller contribution of Fatox to energy expenditure) in patients with NAFLD activity score (NAS) ≥5 vs <5 (p=0.008). Both results were independent of VAT, % body fat and BMI. Compared with the lean control group, patients with NAFLD had lower basal whole-body Fatox (1.2±0.3 vs 1.5±0.4 mg/kgFFM/min, p=0.024) and lower basal hepatic Fatox (ie, β-hydroxybutyrate, p=0.004). During exercise, they achieved lower maximal Fatox (2.5±1.4 vs. 5.8±3.7 mg/kgFFM/min, p=0.002) and lower (p<0.001) than controls. Fatox during exercise was not associated with disease severity (p=0.79). Conclusions Overweight/obese patients with NAFLD had reduced hepatic Fatox and reduced whole-body Fatox under basal and exercise conditions. There was an inverse relationship between ability to oxidise fat in basal conditions and histological features of NAFLD including severity of steatosis and NAS

Comparative study on the protective effects of Yinchenhao Decoction against liver injury induced by alpha-naphthylisothiocyanate and carbon tetrachloride

OBJECTIVE: To optimize the animal model of liver injury that can properly represent the pathological characteristics of dampness-heat jaundice syndrome of traditional Chinese medicine. METHODS: The liver injury in the model rat was induced by alpha-naphthylisothiocyanate (ANIT) and carbon tetrachloride (CCl(4) ) respectively, and the effects of Yinchenhao Decoction (, YCHD), a proved effective Chinese medical formula for treating the dampness-heat jaundice syndrome in clinic, on the two liver injury models were evaluated by analyzing the serum level of alanine aminotransferase (ALT), asparate aminotransferase (AST), alkaline phosphatase (ALP), malondialchehyche (MDA), total bilirubin (T-BIL), superoxide dismutase (SOD), glutathione peroxidase (GSH-PX) as well as the ratio of liver weight to body weight. The experimental data were analyzed by principal component analytical method of pattern recognition. RESULTS: The ratio of liver weight to body weight was significantly elevated in the ANIT and CCl(4) groups when compared with that in the normal control (P<0.01). The contents of ALT and T-BIL were significantly higher in the ANIT group than in the normal control (P<0.05,P<0.01), and the levels of AST, ALT and ALP were significantly elevated in CCl(4) group relative to those in the normal control P<0.01). In the YCHD group, the increase in AST, ALT and ALP levels was significantly reduced (P<0.05, P<0.01), but with no significant increase in serum T-BIL. In the CCl(4) intoxicated group, the MDA content was significantly increased and SOD, GSH-PX activities decreased significantly compared with those in the normal control group, respectively (P<0.01). The increase in MDA induced by CCl(4) was significantly reduced by YCHD P<0.05). CONCLUSION: YCHD showed significant effects on preventing liver injury progression induced by CCl(4), and the closest or most suitable animal model for damp-heat jaundice syndrome may be the one induced by CCl(4).

Epidemiological trend of hepatitis B, liver cirrhosis and liver cancer during 1990 - 2007 in Shandong province, China [山东省乙型肝炎、肝硬化及肝癌流行趋势分析]

Objective To analyze the epidemiological trend of hepatitis B, liver cirrhosis and liver cancer during 1990 to 2007 in Shandong province, and to evaluate the effectiveness of hepatitis B prevention and control measures, so as to provide evidence for policy-making. Methods Based on the routine incidence data of hepatitis B, mortality data of hepatitis B, liver cirrhosis, liver cancer and demographic data, the incidence rate, mortality rate and age-specific mortality rate were calculated and analyzed with simple linear regression model. Results A total of 437094 cases of hepatitis B were reported during 1990 - 2007 with an average yearly morbidity of 27.32 per 100 000 persons and a decreased trend for the 0-9 years old children. At the same time, the adjusted mortality rate for hepatitis B and liver cirrhosis showed a decreased trend and the combined mortality rate decreased from 17.55 /100 000 in 1990 to 4.01 /100 000 in 2007. The mortality of liver cancer was stable during this time (P = 0. 9998). Conclusion Immunization of hepatitis B vaccine may have lowered the incidence of hepatitis B in the target population and the overall mortality rates of liver cirrhosis and liver cancer. Abstract in Chinese 目的 了解山东省1990～2007年乙肝、肝硬化和肝癌的流行状况及变化趋势,初步评价乙肝预防控制措施的效果,为今后防治决策制定提供参考. 方法 根据报告的乙肝发病资料和乙肝、肝硬化、肝癌死亡资料以及历年人口资料,利用发病率、死亡率、年龄别死亡率等指标对上述3种疾病进行流行趋势的分析,并建立简单线性回归模型进行统计分析. 结果 1990～2007年山东省共报告乙肝病例437 094例,年均总发病率为27.32/10万,并呈上升趋势,而0～9岁年龄组的发病率呈显著下降趋势.乙肝和肝硬化调整死亡率下降趋势明显,两者合并死亡率由1990年的17.55/10万下降到2007年的4.01/10万.肝癌调整死亡率基本稳定(P=0.999 8). 结论 做好乙肝疫苗的免疫接种不仅可降低目标人群乙肝的发病,并将最终降低与此相关的肝硬化和肝癌的死亡率.

Dynamic contrast-enhanced magnetic resonance imaging as a biomarker for the pharmacological response of PTK787/ZK 222584, an inhibitor of the vascular endothelial growth factor receptor tyrosine kinases, in patients with advanced colorectal cancer and liver metastases: Results from two phase I studies

Purpose: PTK787/ZK 222584 (PTK/ZK), an orally active inhibitor of vascular endothelial growth factor (VEGF) receptor tyrosine kinases, inhibits VEGF-mediated angiogenesis. The pharmacodynamic effects of PTK/ZK were evaluated by assessing changes in contrast-enhancement parameters of metastatic liver lesions using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) in patients with advanced colorectal cancer treated in two ongoing, dose-escalating phase I studies. Patients and Methods: Twenty-six patients had DCE-MRI performed at baseline, day 2, and at the end of each 28-day cycle. Doses of oral PTK/ZK ranged from 50 to 2000 mg once daily. Tumor permeability and vascularity were assessed by calculating the bidirectional transfer constant (Ki). The percentage of baseline Ki (% of baseline Ki) at each time point was compared with pharmacokinetic and clinical end points. Results: A significant negative correlation exists between the % of baseline Ki and increase in PTK/ZK oral dose and plasma levels (P = .01 for oral dose; P = .0001 for area under the plasma concentration curve at day 2). Patients with a best response of stable disease had a significantly greater reduction in Ki at both day 2 and at the end of cycle 1 compared with progressors (mean difference in % of baseline Ki, 47%, P = .004%; and 51%, P = .006; respectively). The difference in % of baseline Ki remained statistically significant after adjusting for baseline WHO performance status. Conclusion: These findings should help to define a biologically active dose of PTK/ZK. These results suggest that DCE-MRI may be a useful biomarker for defining the pharmacological response and dose of angiogenesis inhibitiors, such as PTK/ZK, for further clinical development. © 2003 by American Society of Clinical Oncology.

Reversibility of liver failure secondary to metastatic breast cancer by vinorelbine and cisplatin chemotherapy

Purpose: The development of liver metastases from breast cancer is associated with a very poor prognosis, estimated at 4 months median survival. Since treatment with many chemotherapeutic agents is relatively contraindicated, we assessed the safety, tolerability and potential efficacy of combination chemotherapy with vinorelbine and cisplatin (ViP). Method: Pilot study in 11 patients with histologically confirmed breast carcinoma, radiological evidence of liver metastases and serum bilirubin greater than 1.5 times the upper limit of normal. Patients received up to six cycles of cisplatin (75 mg/m 2) every 21 days and vinorelbine (20 mg/m 2) on days 1 and 8 of every 21-day cycle. Measurement of liver lesions was performed on CT scan every 8 weeks into treatment. Results: The most frequently reported adverse event was myelosuppression. Other adverse effects included nausea, vomiting and mild neurotoxicity. Two patients died after one treatment with ViP, one of whom suffered an intracerebral haemorrhage that was possibly treatment-related. Improvement in liver function tests was observed in 10 patients, and mean time to normalization of bilirubin levels was 36 days. Partial responses were documented radiologically in 7 out of 11 patients treated. Median overall survival from trial entry was 6.5 months (range 11-364 days), with one patient alive 13 months from trial entry. Conclusion: Normalization of liver function is possible with ViP treatment of metastatic breast cancer, offering the potential to prolong survival. Phase II clinical trials of this regimen in this patient group should include measurement of quality of life in order to assess risk versus benefit.