Quantitative measurement of ultrasound attenuation and Hepato-Renal Index in Non-Alcoholic Fatty Liver Disease.

OBJECTIVE: The aim of this study was to non-invasively explore new methods of ultrasound attenuation measurements in livers of patients with Non-Alcoholic-Fatty-Liver-Disease (NAFLD) and to measure the liver tissue elasticity. MATERIAL AND METHOD: Sixteen patients with NAFLD, twelve patients with liver fibrosis and fifteen healthy subjects were included. Echo Levels (ELs) in dB were measured at 2 and 7 cm depths in the right liver to calculate the attenuation. ELs were measured in liver and right kidney tissue to calculate the Hepato-Renal Index (HRI). This index was calculated both as a difference, HRI-diff; (EL Liver -EL Kidney) and HRI-ratio; (EL Liver / EL Kidney) using built-in software of the ultrasound scanner. Liver tissue elasticity was measured using transient elastography (TE, Fibroscan®). NAFLD and liver fibrosis were confirmed by liver biopsy. RESULTS: We found that HRI- diff was significantly higher in the NAFLD group compared with healthy subjects, 6.2 dB (0.8-11.4) vs.1. 9 dB (0.0-6.1), p=0.012. HRI- ratio was significantly lower between the same two groups, 0.9 dB (0.8-1.02) vs.1.01 dB (0.9-1.12), and p<0.0001. TE, ELs and liver size showed significant differences between NAFLD patients and healthy controls. Between patients with fibrosis and NAFLD the differences were significant for TE, liver size and attenuation. Intra- and interobserver correlation and agreement of ELs were good. CONCLUSION: Measurements of liver tissue using HR-Indexes, ultrasound attenuation, and tissue elasticity may be useful methods to differentiate objectively between steatosis and healthy and quantify the differences.

Combining transient elastography with FIB4 enhances sensitivity in detecting advanced fibrosis of the liver.

OBJECTIVE: To evaluate the predictive value of transient elastography (TE), easy available biochemical scores and a combination of these to detect advanced liver fibrosis (i.e. fibrosis stage ≥F3) in patients with chronic liver disease of different etiologies. MATERIAL AND METHODS: A valid TE was obtained in 418 patients with chronic liver disease of mixed etiologies during the period of 2007-2010. Reliable fibrosis staging and biochemical data for calculation of APRI, FIB4, and AST/ALT-ratio (AAR) were available in 187 cases of which 50 had clinical obvious cirrhosis. Logistic regression analyses were performed to investigate if biochemical scores were significant predictors of advanced fibrosis independent of TE. RESULTS: In the whole group, TE correlated significantly with APRI and FIB4 but not with AAR. In patients with TE ≥7 kPa, a new formula combining TE and FIB4 improved the accuracy for detecting advanced fibrosis. Area under the receiver operating characteristic curve (AUROC) and sensitivity for the combined formula was 0.94 and 0.92, respectively, as opposed to 0.90 and 0.87 for TE alone. After exclusion of cases with clinical obvious cirrhosis, only FIB4 was a significant predictor of advanced liver fibrosis independent of TE. A combined formula gave a marginally improved AUROC in this group. CONCLUSIONS: Our findings suggest that the combination of TE and FIB4 is useful in the prediction of advanced fibrosis. The effect of this combination was marginal when only asymptomatic patients were included. Larger studies are needed to see if this effect is statistically significant and to detect possible differences according to etiology.

Fetuin A in nonalcoholic fatty liver disease: in vivo and in vitro studies.

OBJECTIVE: Fetuin A has been associated with insulin resistance and the metabolic syndrome. We therefore explored the role of fetuin A in nonalcoholic fatty liver disease (NAFLD). DESIGN: Cross-sectional and intervention studies. METHODS: We included 111 subjects with histologically proven NAFLD of whom 44 participated in a randomized, controlled trial with metformin. One hundred and thirty-one healthy subjects and 13 subjects undergoing hepatic surgery for metastatic cancer served as controls. Main outcome variables were circulating levels of fetuin A according to the presence of NAFLD, hepatic gene expression of fetuin A and key enzymes in glucose and lipid metabolism, and the effect of metformin on fetuin A levels in vivo and in vitro (HepG2 cells). RESULTS: Fetuin A levels were significantly higher in NAFLD patients compared with controls (324 ± 98 vs 225 ± 75 mg/l, P<0.001). NAFLD was a significant predictor of elevated fetuin A levels (ß=174 (95% confidence interval: 110-234)) independent of body mass index, age, sex, fasting glucose, and triglycerides. Hepatic fetuin A mRNA levels correlated significantly with hepatic mRNA levels of key enzymes in lipid (sterol regulatory element-binding protein 1c, carnitine palmitoyltransferase 1) and glucose (phosphoenol pyruvate kinase 1, glucose-6-phosphatase) metabolism. Plasma fetuin A levels decreased significantly after metformin treatment compared with placebo (-40 ± 47 vs 15 ± 82 mg/l, P = 0.008). Metformin induced a dose-dependent decrease in fetuin A secretion in vitro. CONCLUSIONS: Fetuin A levels were elevated in NAFLD. Hepatic expression of fetuin A correlated with key enzymes in glucose and lipid metabolism. Metformin decreased fetuin A levels in vitro.

Intracellular nicotinamide phosphoribosyltransferase protects against hepatocyte apoptosis and is down-regulated in nonalcoholic fatty liver disease.

CONTEXT: Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease in Western and non-Western countries, but its pathogenesis is not fully understood. OBJECTIVE: Based on the role of nicotinamide phosphoribosyltransferase (NAMPT) in fat and glucose metabolism and cell survival, we hypothesized a role for NAMPT/visfatin in the pathogenesis of NAFLD-related disease. DESIGN AND SETTING: We conducted clinical studies at a referral medical center in well-characterized NAFLD patients (n = 58) and healthy controls (n = 27). In addition we performed experimental in vitro studies in hepatocytes. MAIN OUTCOME MEASURES: We examined 1) the hepatic and systemic expression of NAMPT/visfatin in patients with NAFLD and control subjects, 2) the hepatic regulation of NAMPT/visfatin, and 3) the effect of NAMPT/visfatin on hepatocyte apoptosis. RESULTS: Our main findings were as follows. 1) Patients with NAFLD had decreased NAMPT/visfatin expression both systemically in serum and within the hepatic tissue, with no difference between simple steatosis and nonalcoholic steatohepatitis. 2) By studying the hepatic regulation of NAMPT/visfatin in wild-type and peroxisome proliferators-activated receptor (PPAR)alpha(-/-) mice as well as in hepatocytes, we showed that PPARalpha activation and glucose may be involved in the down-regulation of hepatic NAMPT/visfatin expression in NAFLD. 4) Within the liver, NAMPT/visfatin was located to hepatocytes, and our in vitro studies showed that NAMPT/visfatin exerts antiapoptotic effects in these cells, involving enzymatic synthesis of nicotinamide adenine dinucleotide. CONCLUSION: Based on these findings, we suggest a role for decreased NAMPT/visfatin levels in hepatocyte apoptosis in NAFLD-related disease.

Metformin in patients with non-alcoholic fatty liver disease: a randomized, controlled trial.

OBJECTIVE: The antidiabetic agent metformin is regularly discussed as a promising treatment for non-alcoholic fatty liver disease (NAFLD), which is characterized by insulin resistance. However, the evidence for its beneficial effects is limited, and conflicting reports have been published. The purpose of this study was to conduct a randomized, double-blind, placebo-controlled trial to test whether metformin improves liver histology in patients with non-alcoholic fatty liver disease. MATERIAL AND METHODS: Forty-eight patients with biopsy-proven NAFLD were randomized to treatment with metformin (n=24) or placebo (n=24) for 6 months. A second liver biopsy was obtained in all subjects who completed the trial (n=44). Data analyses are restricted to this group (per-protocol analyses). The primary outcome was changes in histologically assessed liver steatosis. Secondary outcomes were changes in NAFLD activity (NAS)-score, liver steatosis assessed by computed tomography (CT), liver transaminases, body-weight, metabolic variables and inflammatory markers. RESULTS: No significant differences between treatment with metformin or placebo were observed for changes in liver steatosis, assessed either histologically or by CT, NAS-score, liver transaminases or on markers of insulin resistance or inflammation. In contrast, beneficial effects of metformin were observed on changes in body-weight (p<0.001), serum levels of cholesterol (p=0.004), LDL-cholesterol (p<0.001), glucose (p=0.032) and on HbA1c (p=0.020). CONCLUSIONS: Treatment with metformin for 6 months was no better than placebo in terms of improvement in liver histology in patients with NAFLD. Nevertheless, the use of metformin could still be beneficial in this group as it is associated with a reduction in serum levels of lipids and glucose. (ClinicalTrials.gov number, NCT00303537).

A complex role of activin A in non-alcoholic fatty liver disease.

OBJECTIVES: Recent studies suggest that activin A, a member of the transforming growth factor (TGF) superfamily, is involved in the pathogenesis of liver disorders. We sought to explore its possible role in non-alcoholic fatty liver disease (NAFLD). METHODS: Serum levels of activin A and its natural inhibitor, follistatin, were measured in patients with NAFLD (n=70) and in control subjects (n=30). Gene expression was quantified in liver biopsies obtained from patients with NAFLD (n=13) and controls (n=6). Effects of activin A were examined in Huh7 (human hepatoma cell line) hepatocytes. RESULTS: Patients with NAFLD had significantly elevated serum levels of activin A and follistatin compared with healthy controls. In patients with non-alcoholic steatohepatitis (NASH, n=38), there were particularly high levels of activin A that were significantly related to the degree of hepatic fibrosis. Liver biopsies from NAFLD patients showed a markedly increased activin A-follistatin mRNA ratio, indicating increased hepatic activin A activity. In hepatocytes, activin A enhanced the expression of collagen and TGF-beta(1), promoted matrix metalloproteinase activity, induced mitochondrial beta-oxidation, downregulated fatty acid (FA) synthase activity, promoted decreased weight percentage of saturated FAs, and altered the composition of polyunsaturated FAs. CONCLUSIONS: Our findings support the complex role of activin A in the pathogenesis of NAFLD, involving effects on fibrosis and lipid accumulation.

Gene expression profiles reflect sclerosing cholangitis activity in abcb4 (-/-) mice.

OBJECTIVE: Abcb4 (-/-) mice secrete phosphatidylcholine-deficient bile and develop sclerosing cholangitis (SC), a condition that involves differential hepatic transcription of genes governing inflammation, tissue remodelling and fibrosis. The objective of this study was to test the hypothesis that genes involved in the regulation of tissue inflammation and fibrosis display transcription rates that parallel differences in abcb4 (-/-) SC activity. The activity of abcb4 (-/-) SC can be altered through dietary intervention: abcb4 (-/-) mice fed cholic acid (CA) display high SC activity, whereas ursodeoxycholic acid (UDCA)-fed mice display low SC activity. MATERIAL AND METHODS: Differential hepatic transcription of genes was measured in abcb4 (-/-) mice maintained on CA- and UDCA-supplemented diets using cDNA microarrays. Abcb4 (+/+) mice served as controls. Differential transcription of selected genes was verified by real-time polymerase chain reaction. Liver tissue pathology was quantified by histopathology scoring. RESULT: Histopathology score, reflecting increased inflammation and fibrosis, was increased in CA-fed mice compared with UDCA-fed mice. cDNA microarray analysis showed up-regulation of 1582 genes in livers of CA-fed mice in contrast to 573 genes in UDCA-fed mice. Differential transcription of Ccl2, Ccl20, Cxcl10, Nfkappab1, Nfkappab2, Tgfbeta1, Tgfbeta2, Sparc, Ctgf, Lgals3, Elf3, Spp1, Pdgfa, Pdgfrb, Col1a1, Col1a2 and Col4a1 genes paralleled the unequal SC activities of CA- and UDCA-fed abcb4 (-/-) mice. CONCLUSIONS: The numbers of differentially transcribed genes and the transcriptional activity of genes relating to inflammation, tissue remodelling and fibrosis parallel disease activity in CA- and UDCA-fed abcb4 (-/-) mice harbouring SC. Data on their hepatic transcription can gauge SC disease activity.

Multiple inflammatory-, tissue remodelling- and fibrosis genes are differentially transcribed in the livers of Abcb4 (-/ - ) mice harbouring chronic cholangitis.

OBJECTIVE: Abcb4 (-/-) mice secrete phosphatidylcholine-free, cytotoxic bile and develop chronic cholangitis. The aim of this study was to identify differentially transcribed genes whose products contribute to the liver tissue pathology during this disease. MATERIAL AND METHODS: Hepatic gene transcription was measured in 3-, 6-, 9- and 20-week-old Abcb4 (-/-) mice (FVB.129P2-abcb4(tm1Bor)/J) using cDNA microarrays, with FVB/NJ Abcb4 (+/+) mice serving as controls. Focus was on inflammatory-, remodelling- and fibrosis genes. Marked differential transcription of inflammatory-, tissue remodelling- and fibrosis genes found by cDNA microarrays was verified by real-time polymerase chain reaction (PCR). Liver pathology was quantified by histopathology scoring. RESULTS: Transcription of clade A3 Serpin genes showed early, marked down-regulation. The chemokine genes Ccl2, Ccl20 and Cxcl10 were markedly up-regulated. Tissue remodelling- and fibrosis genes exhibiting markedly up-regulated transcription included: Ctgf, Elf3, Lgals3, Mmp12, Mmp15, Spp1, Loxl2, Pdgfa, Pdgfrb, Sparc, Tgfb1, Tgfb2, Tgfbi, Tgfbr2 and Col1a1, Col1a2, Col2a1, Col3a1, Col4a1 genes. Microarray-based recordings of differential gene transcription of the majority of these genes harmonized with the liver histopathology score. Thus, cDNA microarray-based analysis showed increasing differential transcription of several inflammatory-, tissue remodelling- and fibrosis genes during the first 9 weeks of disease and a tendency towards differential transcription to stabilize at an elevated level from 9 to 20 weeks of disease. CONCLUSIONS: Multiple genes regulating inflammation, tissue remodelling and fibrosis not previously linked to Abcb4 (-/-) cholangitis are identified as being differentially transcribed in Abcb4 (-/-) livers, where they contribute to the pathogenesis of liver tissue pathology.

Systemic inflammation in nonalcoholic fatty liver disease is characterized by elevated levels of CCL2.

BACKGROUND/AIMS: To elucidate the role of systemic inflammation in nonalcoholic fatty liver disease (NAFLD). METHODS: Serum samples in 47 patients with histologically verified NAFLD (22 with simple steatosis and 25 with nonalcoholic steatohepatitis [NASH]), and in 30 age-, sex- and ethnicity-matched healthy controls, were assessed for (i) general markers of inflammation (C-reactive protein [CRP], tumor necrosis factor [TNF]-alpha, and interleukin [IL]-6), (ii) chemokines (CC-chemokine ligand [CCL] 2/monocyte chemoattractant protein [MCP]-1, CCL19 and CCL21), (iii) adipocytokines related to insulin resistance and inflammation (adiponectin and leptin) and (iv) a marker of oxidative stress (8-isoprostane-F2alpha). RESULTS: Serum levels of several inflammatory cytokines were increased in NAFLD as compared to controls, and IL-6 (P=0.017), CCL2/MCP-1 (P=0.008) and CCL19 (P=0.001), but not CRP (P=0.199), remained elevated also after correction for sex, body mass index (BMI) and age. Comparing NASH with simple steatosis, levels of TNF-alpha (P=0.024) and CCL2/MCP-1 (P=0.012) were elevated and adiponectin (in women) (P=0.001) were decreased also after adjustment for sex, BMI and presence of the metabolic syndrome. CONCLUSIONS: Our results indicate that patients with NAFLD are characterized by a low-grade systemic inflammation. The high CCL2/MCP-1 levels in NASH might be of importance for the conversion from simple steatosis to NASH.

Abnormal glucose tolerance is a predictor of steatohepatitis and fibrosis in patients with non-alcoholic fatty liver disease.

OBJECTIVE: The majority of patients with non-alcoholic fatty liver disease (NAFLD) have simple steatosis. A minority, however, present with non-alcoholic steatohepatitis (NASH), a condition that can lead to advanced fibrosis and cirrhosis. The frequencies of NASH and fibrosis among patients with NAFLD and sustained elevation of liver function tests (LFT) are uncertain. Our aim was to estimate these frequencies. We characterize a population with NAFLD, with special emphasis on insulin resistance and the metabolic syndrome, and study possible predictors for different stages of the disease. MATERIAL AND METHODS: All referred patients with sustained elevation of LFT, radiological evidence or clinical suspicion of fatty liver, and absence of other liver disease, were invited to participate in our study in the period June 2002 to December 2004. RESULTS: Of 129 patients who met the inclusion criteria, 88 underwent liver biopsy. NAFLD was verified in 83 of them. Among these patients, 59 (71%) had the metabolic syndrome, 41 (49%) had NASH and 36 (43%) had fibrosis. Abnormal glucose tolerance (T2DM or impaired glucose tolerance) was the only independent risk factor for NASH (OR: 3.14; 95% CI: 1.20-8.23). Independent predictors for fibrosis were abnormal glucose tolerance (OR: 3.83; 95% CI: 1.29-11.40) and body mass index (OR: 1.20; 95% CI: 1.06-1.36) per kg/m2. CONCLUSIONS: Both NASH and fibrosis are frequently present among patients with NAFLD and sustained elevation of LFT. The probability of these potentially progressive stages of NAFLD increases with the presence of abnormal glucose tolerance.