Bryan D. Maliken

Reduced adiponectin signaling due to weight gain results in nonalcoholic steatohepatitis through impaired mitochondrial biogenesis

Obesity and adiponectin depletion have been associated with the occurrence of nonalcoholic fatty liver disease (NAFLD). The goal of this study was to identify the relationship between weight gain, adiponectin signaling, and development of nonalcoholic steatohepatitis (NASH) in an obese, diabetic mouse model. Leptin‐receptor deficient (Leprdb/db) and C57BL/6 mice were administered a diet high in unsaturated fat (HF) (61%) or normal chow for 5 or 10 weeks. Liver histology was evaluated using steatosis, inflammation, and ballooning scores. Serum, adipose tissue, and liver were analyzed for changes in metabolic parameters, messenger RNA (mRNA), and protein levels. Leprdb/db HF mice developed marked obesity, hepatic steatosis, and more than 50% progressed to NASH at each timepoint. Serum adiponectin level demonstrated a strong inverse relationship with body mass (r = −0.82; P < 0.0001) and adiponectin level was an independent predictor of NASH (13.6 μg/mL; P < 0.05; area under the receiver operating curve (AUROC) = 0.84). White adipose tissue of NASH mice was characterized by increased expression of genes linked to oxidative stress, macrophage infiltration, reduced adiponectin, and impaired lipid metabolism. HF lepr db/db NASH mice exhibited diminished hepatic adiponectin signaling evidenced by reduced levels of adiponectin receptor‐2, inactivation of adenosine monophosphate activated protein kinase (AMPK), and decreased expression of genes involved in mitochondrial biogenesis and β‐oxidation (Cox4, Nrf1, Pgc1α, Pgc1β and Tfam). In contrast, recombinant adiponectin administration up‐regulated the expression of mitochondrial genes in AML‐12 hepatocytes, with or without lipid‐loading. Conclusion: Leprdb/db mice fed a diet high in unsaturated fat develop weight gain and NASH through adiponectin depletion, which is associated with adipose tissue inflammation and hepatic mitochondrial dysfunction. We propose that this murine model of NASH may provide novel insights into the mechanism for development of human NASH. (Hepatology 2014;60:133–145)

Hepatic reticuloendothelial system cell iron deposition is associated with increased apoptosis in nonalcoholic fatty liver disease

The aim of this study was to examine the relationship between the presence of hepatic iron deposition, apoptosis, histologic features, and serum markers of oxidative stress (OS) and cell death in nonalcoholic fatty liver disease (NAFLD). Clinical, biochemical, metabolic, and independent histopathologic assessment was conducted in 83 unselected patients with biopsy‐proven NAFLD from a single center. Apoptosis and necrosis in serum was quantified using serum cytokeratin 18 (CK18) M30 and M65 enzyme‐linked immunosorbent assays and in liver by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining in situ. Serum malondialdehyde (MDA) and thioredoxin‐1 (Trx1) levels were measured to evaluate OS. Presence of reticuloendothelial system (RES) cell iron in the liver was associated with nonalcoholic steatohepatitis (P < 0.05) and increased hepatic TUNEL staining (P = 0.02), as well as increased serum levels of apoptosis‐specific (M30; P = 0.013) and total (M65; P = 0.006) CK18 fragments, higher MDA (P = 0.002) and lower antioxidant Trx1 levels (P = 0.012), compared to patients without stainable hepatic iron. NAFLD patients with a hepatocellular (HC) iron staining pattern also had increased serum MDA (P = 0.006), but not M30 CK18 levels or TUNEL staining, compared to subjects without stainable hepatic iron. Patients with iron deposition limited to hepatocytes had a lower proportion of apoptosis‐specific M30 fragments relative to total M65 CK18 levels (37% versus ≤25%; P < 0.05). Conclusions: Presence of iron in liver RES cells is associated with NASH, increased apoptosis, and increased OS. HC iron deposition in NAFLD is also associated with OS and may promote hepatocyte necrosis in this disease. (HEPATOLOGY 2013)

Differences in hepatic expression of iron, inflammation and stress-related genes in patients with nonalcoholic steatohepatitis.

Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease worldwide. We have previously shown that hepatic reticuloendothelial system (RES) iron deposition is associated with an advanced degree of nonalcoholic steatohepatitis (NASH) in humans. In this study, we aimed to determine differentially expressed genes related to iron overload, inflammation and oxidative stress pathways, with the goal of identifying factors associated with NASH progression. Seventy five patients with NAFLD were evaluated for their biochemical parameters and their liver tissue analyzed for NASH histological characteristics. Gene expression analysis of pathways related to iron homeostasis, inflammation and oxidative stress was performed using real-time PCR. Gene expression was compared between subjects based on disease status and presence of hepatic iron staining. We observed increased gene expression of hepcidin (HAMP) (2.3 fold, p = 0.027), transmembrane serine proteinase 6 (TMPRSS6) (8.4 fold, p = 0.003), signal transducer and activator of transcription 3 (STAT3) (5.5 fold, p = 0.004), proinflammatory cytokines; IL-1β (2.7 fold, p = 0.046) and TNF-α (3.8 fold, p = 0.001) in patients with NASH. TMPRSS6, a negative regulator of HAMP, is overexpressed in patients with NASH and HIF1α (hypoxia inducible factor-1) is downregulated. NAFLD patients with hepatic iron deposition exhibited higher hepci-din expression (3.1 fold, p = 0.04) but lower expression of cytokines. In conclusion, we observed elevated hepatic HAMP expression in patients with NASH and in NAFLD patients who had hepatic iron deposition, while proinflammatory cytokines displayed elevated expression only in patients with NASH, suggesting a regulatory role for hepcidin in NAFL to NASH transition and in mitigating inflammatory responses.

S1845 Combining Genetic and Dietary Factors in Creation of a Novel Model of Nonalcoholic Steatohepatitis (NASH) in Mice

Aim: To characterize hepatocyte apoptosis in a nutritional model of steatohepatitis induced by modified atherogenic diet (M-Ath). Methods: Liver tissue samples were obtained at sacrifice from 5 animals belonging to 4 treatment groups (control, fructose, atherogenic, and M-Ath). Hepatocyte apoptosis in liver sections was quantified by counting the number of TUNEL positive cells in 10 random microscopic fields (x 40). Caspase 3 activity was measured in liver tissue lysate following 19 hr incubation. Results: Compared to controls, the number of TUNEL positive hepatocytes was not different in atherogenic and M-Ath diet groups, but the fructose group had significantly lower number of TUNEL positive hepatocytes. Pigs in the M-Ath diet had significantly higher Caspase 3 activity when compared to the other three groups. There was no detectable Caspase 3 activity in liver lysate of the fructose group pigs. Serum ALT values were not different among control, atherogenic and M-Ath diets but the fructose group had significantly lower ALT values. Conclusions: Feeding Ossabaw pigs a modified atherogenic diet resulted in the development of abnormal liver histology associated with increased caspase 3 activity as a marker of hepatocyte apoptosis. These results identify a relevant experimental model for studies to understand the pathophysiology of NASH development as well as to test novel treatment strategies for patients with this condition.