Synergistic steatohepatitis by moderate obesity and alcohol in mice despite increased adiponectin and p-AMPK

Abstract

Background & aims: Mechanisms underlying synergistic liver injury caused by alcohol and obesity are not clear. We have produced a mouse model of synergistic steatohepatitis by recapitulating the natural history of the synergism seen in patients for mechanistic studies.

Methods: Moderate obesity was induced in mice by 170% overnutrition in calories using intragastric overfeeding of high fat diet. Alcohol (low or high dose) was then co-administrated to determine its effects.

Results: Moderate obesity plus alcohol intake causes synergistic steatohepatitis in an alcohol dose-dependent manner. A heightened synergism is observed when a high alcohol dose (32g/kg/d) is used, resulting in plasma ALT reaching 392±28U/L, severe steatohepatitis with pericellular fibrosis, marked M1 macrophage activation, a 40-fold induction of iNos, and intensified nitrosative stress in the liver. Hepatic expression of genes for mitochondrial biogenesis and metabolism are significantly downregulated, and hepatic ATP level is decreased. Synergistic ER stress evident by elevated XBP-1, GRP78 and CHOP is accompanied by hyperhomocysteinemia. Despite increased caspase 3/7 cleavage, their activities are decreased in a redox-dependent manner. Neither increased PARP cleavage nor TUNEL positive hepatocytes are found, suggesting a shift of apoptosis to necrosis. Surprisingly, the synergism mice have increased plasma adiponectin and hepatic p-AMPK, but adiponectin resistance is shown downstream of p-AMPK.

Conclusions: Nitrosative stress mediated by M1 macrophage activation, adiponectin resistance, and accentuated ER and mitochondrial stress underlie potential mechanisms for synergistic steatohepatitis caused by moderate obesity and alcohol.

Figure 1. Overfeeding plus alcohol induces synergistic steatohepatitis

(A) Schematic presentation of concomitant OF and alcohol infusion. The caloric intake in the overfed mice were gradually increased from initial 580 Cal/kg/day to 986 Cal/kg/day on day 17 and kept to the end. On day 17, alcohol was added and gradually increased to a final alcohol dose of 23g/kg/day or 32g/kg/day. Shaded area represents the amount of calories derived from alcohol in daily caloric intake. (B) Plasma ALT levels, n=5 for each group. (C) Liver H&E, arrowhead indicates mononuclear cell infiltration; and reticulin staining of fibrosis. ALT, alanine aminotransferase; OF, overfeeding; Alc, alcohol; Reti, reticulin.

Figure 2. Adiponectin signaling in overfeeding plus alcohol mice

(A) Plasma adiponectin levels, n=5 for each group. (B) Hepatic mRNA levels of AdipoR1 & 2 by qRT-PCR, n=5 for each group. (C) Immunoblot of adiponectin effectors involved in lipid metabolism. * p < 0.05 compared to control; † p < 0.05 compared to alcohol only group. OF, overfeeding; Alc, alcohol; AdipoR, adiponectin receptor.

Figure 3. Overfeeding and alcohol enhance ER stress and activate apoptotic pathway

Immunoblot for (A) ER stress markers eIF2α, GRP78, and XBP-1; (B) cell death signaling molecules JNK 1&2 (arrow, p-JNK1) and survival signaling molecules ERK 1&2; and (C) apoptotic executioner caspase-3 & -7; CHOP, a mediator of ER-induced apoptosis; and PARP, a downstream target of caspase-3 & -7. * p < 0.05 compared to control; † p < 0.05 compared to respective alcohol groups. (D) qRT-PCR analyses of anti-apoptotic genes Bcl-2, Bcl-xL, pro-apoptotic genes Bad, Bax and Bim, as well as Chop. n =5 for each group. * p < 0.05 compared to control; † p < 0.05 compared to respective alcohol group. (E) Hepatic caspase 3 & 7 activities were expressed as percent values relative to the Control’s, which was not preincubated with DTT (the 3^rd open bar from left). DTT preincubation is shown in black bar. Livers from mice injected with PBS (n=2) and Jo2 antibody (n=2) serve as negative and positive controls of apoptosis, respectively. n= 5–8 for each experimental groups. * p < 0.05 compared to their respective Control groups.

Figure 4. Effect of overfeeding and alcohol on hepatic mitochondrial biogenesis and ATP production

(A) qRT-PCR analysis of hepatic genes involved in mitochondrial biogenesis (PGC-1a, Nrf-1, TFB1M, TFB2M) and metabolism (PPARa,, CoxII, Cyto C), as well as peroxisomal fatty acid oxidation (Acox1). * P < 0.05 and ** P < 0.01 compared to Control; † P < 0.05 compared to respective alcohol group, n = 5 for each group. (B) Hepatic ATP contents, n= 5–8 for each groups.

Figure 5. Over feeding and alcohol result in synergistic macrophage infiltration in the liver and white adipose tissue (WAT), with contrasting M1 activation in the liver and M2 activation in WAT

(A) and (B) qRT-PCR analyses of macrophage marker (CD68), M1 markers (iNOS, TNFa), M2 marker (Arg1, Il-10), and adiponectin. (C) H&E stainging of WAT. (D) immunostaining of macrophage marker CD68. Arrowheads in C and D indicate macrophage infiltration. (E) Immunostaining of nitrotyrosine in the liver. The green fluorescence intensity correlates with the degree of tyrosine nitration. * p < 0.05 and ** p < 0.01 compared to control; † p < 0.05 compared to respective alcohol group, n = 5 for each group.