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

UNLABELLED: 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 (Lepr(db/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. Lepr(db/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 ß-oxidation (Cox4, Nrf1, Pgc1α, Pgc1ß and Tfam). In contrast, recombinant adiponectin administration up-regulated the expression of mitochondrial genes in AML-12 hepatocytes, with or without lipid-loading. CONCLUSION: Lepr(db/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.

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

UNLABELLED: 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.

JNK3-mediated apoptotic cell death in primary dopaminergic neurons.

Investigation of mechanisms responsible for dopaminergic neuron death is critical for understanding the pathogenesis of Parkinson's disease, yet this is often quite challenging technically. Here, we describe detailed methods for culturing primary mesencephalic dopaminergic neurons and examining the activation of c-Jun N-terminal protein Kinase (JNK) in these cultures. We utilized immunocytochemistry and computerized analysis to quantify the number of surviving dopaminergic neurons and JNK activation in dopaminergic neurons. TUNEL staining was used to quantify apoptotic cell death. siRNA was used to specifically inhibit JNK3, the neural specific isoform of JNK. Our data implicate the activation of JNK3 in rotenone-induced dopaminergic neuron apoptosis.

Basic fibroblast growth factor protects against rotenone-induced dopaminergic cell death through activation of extracellular signal-regulated kinases 1/2 and phosphatidylinositol-3 kinase pathways.

Administration of rotenone to rats reproduces many features of Parkinson's disease, including dopaminergic neuron degeneration, and provides a useful model to study the pathogenesis of Parkinson's disease. However, the cell death mechanisms induced by rotenone and potential neuroprotective mechanisms against rotenone are not well defined. Here we report that rotenone-induced apoptosis in human dopaminergic SH-SY5Y cells is attenuated by pretreatment with several growth factors, most notably basic fibroblast growth factor (bFGF). bFGF activated both extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphatidylinositol-3 kinase (PI3-kinase) pathways in SH-SY5Y cells. Ectopic activation of ERK1/2 or PI3-kinase protected against rotenone, whereas inhibition of either pathway attenuated bFGF protection. Reducing the expression of the proapoptotic protein Bcl-2-associated death protein (BAD) by small interfering RNA rendered SH-SY5Y cells resistant to rotenone, implicating BAD in rotenone-induced cell death. Interestingly, bFGF induced a long-lasting phosphorylation of BAD at serine 112, suggesting BAD inactivation through the ERK1/2 signaling pathway. Moreover, primary cultured dopaminergic neurons from mesencephalon were more sensitive to rotenone-induced cell death than nondopaminergic neurons in the same culture. The loss of dopaminergic neurons was blocked by bFGF, an inhibition dependent on ERK1/2 and PI3-kinase signaling. These data suggest that rotenone-induced dopaminergic cell death requires BAD and identify bFGF and its activation of ERK1/2 and PI3-kinase signaling pathways as novel intervention strategies to block cell death in the rotenone model of Parkinson's disease.