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907 NAFLD AND T2D: A GENETIC OR METABOLIC ISSUE?
S340
POSTERS
906 FIXATION TEMPERATURE ALTERS INTRAMITOCHONDRIAL CRYSTALS (IMC) IN HUMAN NASH: IMPLICATIONS FOR MITOCHONDRIAL CHANGES IN STEATOHEPATITIS S.H. Caldwell1 , L.A.R. Freitas2 , S.H. Park1 , M.L.V. Moreno2 , C.A. Davis3 , J.A. Redick3 , J.T. Patrie4 , H.P. Cotrim5 , V.M.R. Lima6 , C.K. Argo1 , A.M. Al-Osaimi1 . 1 GI/Hepatology, University of Virginia Medical Center, Charlottesville, VA, USA; 2 Pathology, Oswaldo Cruz Foundation, Salvador-Bahia, Brazil; 3 Advanced Microscopy Center, University of Virginia, Charlottesville, VA, USA; 4 Health Evaluations, University of Virginia, Charlottesville, VA, USA; 5 GI/Hepatology, Universidade Federal Da Bahia, Salvador-Bahia, Brazil; 6 GI/Hepatology, University of S˜ao Paulo School of Medicine, S˜ao Paulo, Brazil E-mail: [email protected];[email protected] We described IMC in human NASH identical to that reported in Wilson Disease and alcoholic steatohepatitis (J Hepatol 1999;31:430). In human NASH, crystals are seen on transmission electron microscopy (TEM) as parallel strands in 5−10% of mitochondria in afflicted cells, occur throughout the lobule and increase with successful thiazolidinedione therapy (Hepatol 2004;39:1423, Hepatol 2007;46:1101). Aim: We hypothesize that IMC represent changes in the cristae lipid bilayer subject to temperature. To test this, we performed a blinded, prospective, quantitative study on crystal detection by TEM. Methods: From NASH patients’ biopsies, three 1−2 mm sections were immediately fixed for two hours (4% paraformaldehyde, 2.5% glutaraldehyde) at a controlled temperature: hot (37C), cold (4C) and room temperature (RT). The specimens were post-fixed in osmium tetroxide and embedded. 4 patients with active NASH were included, resulting in 12 blocks (4 blocks per condition). Two pairs of non-overlapping ultrathin sections (85 nm) were cut from each block, placed on Fullam copper grids and contrast stained resulting in 48 grids (16 grids per condition). Blinded quantitative morphometry was performed by high resolution 12k TEM images with manual counting of the total and crystal containing mitochondria in each area. Results: 549 TEM images of uniform area were manually counted – 187 from hot, 187 from cold and 175 from RT. Unblinding revealed that the percentage of crystal containing mitochondria was 21.7±31 vs 3.81±9.6 vs 7.9±17.9 for hot, cold and RT. Cox Proportional Hazard model showed that the distribution of the percentage of mitochondria with crystals was influenced by temperature (P = 0.001). The percentage of mitochondria with crystals was greater with hot fixation compared to either cold fixation (p < 0.001) or RT (p = 0.007). There was no significant difference between cold and RT (p = 0.249). Conclusion: In this prospective, blinded, quantitative assessment of intramitochondrial crystals in human NASH, fixation temperature strongly influenced the detection of these structures. We speculate that IMC represent conformational changes in the cristae lipid bilayer (hexagonal phase) and may result from the heat producing effects of mitochondrial uncoupling during oxidative stress and associated changes in mitochondrial cation and DNA content. 907 NAFLD AND T2D: A GENETIC OR METABOLIC ISSUE? L. Carulli, S. Rondinella, A. Rudilosso, D. Ganazzi, M. Bertolotti, P. Loria, N. Carulli. Department of Medicine, Endocrinology, Metabolism and Geriatrics, NOCSAE, Modena, Italy E-mail: [email protected] Background: Type 2 diabetes (T2D) seems a risk factor for the development of Non Alcoholic Fatty Liver Disease (NAFLD) and for its progression to fibrosis. The pathogenesis of NAFLD-T2D association is not known. Recent data have shown that hyperinsulinemia and Insulin resistance (IR) may be the primary phenomenon in NAFLD as well as inflammation.
Aim of the study was to evaluate the prevalence of NAFLD in T2D, to correlate NAFLD with the Metabolic Syndrome (MetS) features and with diabetes therapy, to evaluate the relation between NAFLD and genetic polymorphisms associated to IR (PC-1 K121Q) and inflammation (IL-6 −174 C/G). Methods: 80 diabetic subjects were enrolled and underwent ultrasound (US) to establish the presence of bright liver, medical history to ruled out alcohol consumption and other liver diseases aetiology. Steatosis was defined according to standardized ultrasonographic criteria and for each criterion a score was assigned as indicator of fatty liver infiltration (Fatty Liver Indicator, FLI): major criterion, mandatory, was increasing discrepance of echo amplitude between liver and kidney (score 0−3); minor criteria (score 0−1 for each one) were loss of echoes from depth, from hepatic vessels walls, from diaphragm, gallbladder wall blurring, presence of focal sparing areas. Results: Diabetic subjects studied were overweight with BMI = 28.60 (25º−75º = 25.35–32.95), had normal lipid profile and uric acid and had higher GPT levels GOT/GPT = 20/28 U/L (25º–75º; GOT: 7.00–27.25 and GPT: 21.00−38.00). 1. Among diabetic subjects 77.5% had Fatty Liver • FLI did correlate with BMI (p < 0.01), Waist circumference (p < 0.01), Total-cholesterol (p < 0.01) and TG (p < 0.01). BMI and Total-cholesterol resulted to be independent risk factors of steatosis. • No correlation was found between therapy and severity of NAFLD. 2. No significant difference in polymorphisms prevalence was observed in NAFLD subjects versus a control group. Discussion: Our data show that T2D patients have a very high prevalence of NAFLD which is probably related to hyperinsulinism and IR. This is further supported by the positive correlation of NAFLD with BMI, Waist, Tot-cholesterol and TG. The lipogenic effects of insulin may underlie such relationship. NAFLD associates with some features of MetS whereas no significant genetic component is found.
908 NONALCOHOLIC FATTY LIVER DISEASE (NAFLD) IN BRAZIL: CLINICAL AND HISTOLOGICAL PROFILE H.P. Cotrim1 , E.R. Parise2 , N. Leite3 , C. Oliveira4 , A. Martinelli5 , J. Galizzi6 , R.C. Silva7 , A. Mattos8 , L. Pereira9 , W. Amorim10 , C. Ivantes11 , F. Souza12 , M. Costa13 , L. Maia14 , M. Pessoa15 , F. Oliveira16 . 1 Universidade Federal Bahia – Brazil Universidade Federal Da Bahia, Salvador, Brazil; 2 Universidade Federal S˜ao Paulo, S˜ao Paulo, Brazil; 3 Universidade Federal Rio de Janeiro, Rio de Janeiro, Brazil; 4 Universidade S˜ ao Paulo, S˜ao Paulo, Brazil; 5 Universidade S˜ao Paulo, Ribeir˜ao Preto, Brazil; 6 Felicio Rocho Hospital, Universidade Federal Minas Gerais, Minas Gerais, Brazil; 7 Faculdade de Medicina – S˜ao Jos´e Do Rio Preto, S˜ao Paulo, Brazil; 8 Funda¸ca˜ o Faculdade Federal de Ciˆencias M´edicas, Rio Grande do Sul, Brazil; 9 Universidade de Pernambuco, Recife, Brazil; 10 Universidade Federal Da Paraiba, Jo˜ao Pessoa, 11 Universidade Federal Do Parana, Brazil; 12 Universidade Federal Minas Gerais, Brazil; 13 Hospital de Base, Brasilia, DF, Brazil; 14 Santa Casa Belem-Para, Brazil; 15 S˜ ao Paulo, Brazil; 16 Juiz de Fora, Minas Gerais, Brazil E-mail: [email protected] Background: Brazil is a large country and the population in each area may present different characteristics. The prevalence of NAFLD in Brazil is not known, however the relevance of this liver disease in this country have been shown. The present study is the first that aimed to evaluate the clinical and histological profile of NAFLD in Brazil. Methodology: The study included patients with NAFLD around Brazil. Clinical and histological parameters were studied. Individuals with history of alcohol intake, B and C virus infection, haemocromatosis and autoimmune diseases were excluded. Histological diagnosis: steatosis and steatohepatitis (steatosis, ballooning of hepatocytes or fibrosis). Fibrosis was classified into 5 stages (S): S 0, without fibrosis; S 1, fibrosis limited
POSTERS
906 FIXATION TEMPERATURE ALTERS INTRAMITOCHONDRIAL CRYSTALS (IMC) IN HUMAN NASH: IMPLICATIONS FOR MITOCHONDRIAL CHANGES IN STEATOHEPATITIS S.H. Caldwell1 , L.A.R. Freitas2 , S.H. Park1 , M.L.V. Moreno2 , C.A. Davis3 , J.A. Redick3 , J.T. Patrie4 , H.P. Cotrim5 , V.M.R. Lima6 , C.K. Argo1 , A.M. Al-Osaimi1 . 1 GI/Hepatology, University of Virginia Medical Center, Charlottesville, VA, USA; 2 Pathology, Oswaldo Cruz Foundation, Salvador-Bahia, Brazil; 3 Advanced Microscopy Center, University of Virginia, Charlottesville, VA, USA; 4 Health Evaluations, University of Virginia, Charlottesville, VA, USA; 5 GI/Hepatology, Universidade Federal Da Bahia, Salvador-Bahia, Brazil; 6 GI/Hepatology, University of S˜ao Paulo School of Medicine, S˜ao Paulo, Brazil E-mail: [email protected];[email protected] We described IMC in human NASH identical to that reported in Wilson Disease and alcoholic steatohepatitis (J Hepatol 1999;31:430). In human NASH, crystals are seen on transmission electron microscopy (TEM) as parallel strands in 5−10% of mitochondria in afflicted cells, occur throughout the lobule and increase with successful thiazolidinedione therapy (Hepatol 2004;39:1423, Hepatol 2007;46:1101). Aim: We hypothesize that IMC represent changes in the cristae lipid bilayer subject to temperature. To test this, we performed a blinded, prospective, quantitative study on crystal detection by TEM. Methods: From NASH patients’ biopsies, three 1−2 mm sections were immediately fixed for two hours (4% paraformaldehyde, 2.5% glutaraldehyde) at a controlled temperature: hot (37C), cold (4C) and room temperature (RT). The specimens were post-fixed in osmium tetroxide and embedded. 4 patients with active NASH were included, resulting in 12 blocks (4 blocks per condition). Two pairs of non-overlapping ultrathin sections (85 nm) were cut from each block, placed on Fullam copper grids and contrast stained resulting in 48 grids (16 grids per condition). Blinded quantitative morphometry was performed by high resolution 12k TEM images with manual counting of the total and crystal containing mitochondria in each area. Results: 549 TEM images of uniform area were manually counted – 187 from hot, 187 from cold and 175 from RT. Unblinding revealed that the percentage of crystal containing mitochondria was 21.7±31 vs 3.81±9.6 vs 7.9±17.9 for hot, cold and RT. Cox Proportional Hazard model showed that the distribution of the percentage of mitochondria with crystals was influenced by temperature (P = 0.001). The percentage of mitochondria with crystals was greater with hot fixation compared to either cold fixation (p < 0.001) or RT (p = 0.007). There was no significant difference between cold and RT (p = 0.249). Conclusion: In this prospective, blinded, quantitative assessment of intramitochondrial crystals in human NASH, fixation temperature strongly influenced the detection of these structures. We speculate that IMC represent conformational changes in the cristae lipid bilayer (hexagonal phase) and may result from the heat producing effects of mitochondrial uncoupling during oxidative stress and associated changes in mitochondrial cation and DNA content. 907 NAFLD AND T2D: A GENETIC OR METABOLIC ISSUE? L. Carulli, S. Rondinella, A. Rudilosso, D. Ganazzi, M. Bertolotti, P. Loria, N. Carulli. Department of Medicine, Endocrinology, Metabolism and Geriatrics, NOCSAE, Modena, Italy E-mail: [email protected] Background: Type 2 diabetes (T2D) seems a risk factor for the development of Non Alcoholic Fatty Liver Disease (NAFLD) and for its progression to fibrosis. The pathogenesis of NAFLD-T2D association is not known. Recent data have shown that hyperinsulinemia and Insulin resistance (IR) may be the primary phenomenon in NAFLD as well as inflammation.
Aim of the study was to evaluate the prevalence of NAFLD in T2D, to correlate NAFLD with the Metabolic Syndrome (MetS) features and with diabetes therapy, to evaluate the relation between NAFLD and genetic polymorphisms associated to IR (PC-1 K121Q) and inflammation (IL-6 −174 C/G). Methods: 80 diabetic subjects were enrolled and underwent ultrasound (US) to establish the presence of bright liver, medical history to ruled out alcohol consumption and other liver diseases aetiology. Steatosis was defined according to standardized ultrasonographic criteria and for each criterion a score was assigned as indicator of fatty liver infiltration (Fatty Liver Indicator, FLI): major criterion, mandatory, was increasing discrepance of echo amplitude between liver and kidney (score 0−3); minor criteria (score 0−1 for each one) were loss of echoes from depth, from hepatic vessels walls, from diaphragm, gallbladder wall blurring, presence of focal sparing areas. Results: Diabetic subjects studied were overweight with BMI = 28.60 (25º−75º = 25.35–32.95), had normal lipid profile and uric acid and had higher GPT levels GOT/GPT = 20/28 U/L (25º–75º; GOT: 7.00–27.25 and GPT: 21.00−38.00). 1. Among diabetic subjects 77.5% had Fatty Liver • FLI did correlate with BMI (p < 0.01), Waist circumference (p < 0.01), Total-cholesterol (p < 0.01) and TG (p < 0.01). BMI and Total-cholesterol resulted to be independent risk factors of steatosis. • No correlation was found between therapy and severity of NAFLD. 2. No significant difference in polymorphisms prevalence was observed in NAFLD subjects versus a control group. Discussion: Our data show that T2D patients have a very high prevalence of NAFLD which is probably related to hyperinsulinism and IR. This is further supported by the positive correlation of NAFLD with BMI, Waist, Tot-cholesterol and TG. The lipogenic effects of insulin may underlie such relationship. NAFLD associates with some features of MetS whereas no significant genetic component is found.
908 NONALCOHOLIC FATTY LIVER DISEASE (NAFLD) IN BRAZIL: CLINICAL AND HISTOLOGICAL PROFILE H.P. Cotrim1 , E.R. Parise2 , N. Leite3 , C. Oliveira4 , A. Martinelli5 , J. Galizzi6 , R.C. Silva7 , A. Mattos8 , L. Pereira9 , W. Amorim10 , C. Ivantes11 , F. Souza12 , M. Costa13 , L. Maia14 , M. Pessoa15 , F. Oliveira16 . 1 Universidade Federal Bahia – Brazil Universidade Federal Da Bahia, Salvador, Brazil; 2 Universidade Federal S˜ao Paulo, S˜ao Paulo, Brazil; 3 Universidade Federal Rio de Janeiro, Rio de Janeiro, Brazil; 4 Universidade S˜ ao Paulo, S˜ao Paulo, Brazil; 5 Universidade S˜ao Paulo, Ribeir˜ao Preto, Brazil; 6 Felicio Rocho Hospital, Universidade Federal Minas Gerais, Minas Gerais, Brazil; 7 Faculdade de Medicina – S˜ao Jos´e Do Rio Preto, S˜ao Paulo, Brazil; 8 Funda¸ca˜ o Faculdade Federal de Ciˆencias M´edicas, Rio Grande do Sul, Brazil; 9 Universidade de Pernambuco, Recife, Brazil; 10 Universidade Federal Da Paraiba, Jo˜ao Pessoa, 11 Universidade Federal Do Parana, Brazil; 12 Universidade Federal Minas Gerais, Brazil; 13 Hospital de Base, Brasilia, DF, Brazil; 14 Santa Casa Belem-Para, Brazil; 15 S˜ ao Paulo, Brazil; 16 Juiz de Fora, Minas Gerais, Brazil E-mail: [email protected] Background: Brazil is a large country and the population in each area may present different characteristics. The prevalence of NAFLD in Brazil is not known, however the relevance of this liver disease in this country have been shown. The present study is the first that aimed to evaluate the clinical and histological profile of NAFLD in Brazil. Methodology: The study included patients with NAFLD around Brazil. Clinical and histological parameters were studied. Individuals with history of alcohol intake, B and C virus infection, haemocromatosis and autoimmune diseases were excluded. Histological diagnosis: steatosis and steatohepatitis (steatosis, ballooning of hepatocytes or fibrosis). Fibrosis was classified into 5 stages (S): S 0, without fibrosis; S 1, fibrosis limited