The fat-derived hormone adiponectin alleviates alcoholic and nonalcoholic fatty liver diseases in mice (original) (raw)
Chronic ethanol consumption causes significant decrease of circulating adiponectin in mice. Mice fed with a modified high-fat liquid ethanol (LE) diet and pair-fed with high-fat liquid control (LC) diet (25) gained weight throughout the 6-week treatment period. Although the weight gain of LC mice (7.2 ± 0.6 g) was slightly higher, it was not significantly different from that of mice on the LE diet (6.8 ± 0.5 g). Mice fed with the LE diet consumed ethanol at approximately 17–19 g/kg body weight per day. At necropsy, liver-to-body weight ratios in mice receiving ethanol (8.3% ± 0.6%) were significantly higher than those in mice fed with the control diet (6.2% ± 0.4%).
Chronic ethanol consumption caused a significant decrease in circulating concentrations of adiponectin. Plasma adiponectin decreased 32.1% ± 2.9% after 3 weeks and 40.3% ± 4.6% after 4 weeks of feeding with the LE diet (Figure 1a). Decreased adiponectin correlated closely with the development of liver injury, as judged by the plasma levels of ALT activity (Figure 1b). Moreover, there was an inverse relationship between circulating concentrations of adiponectin and TNF-α level following chronic consumption of the LE diet (Figure 1c).
Chronic alcohol consumption decreases plasma adiponectin, increases TNF-α, and induces liver injury. Plasma samples were collected at different stages after mice had been fed with either high-fat LC diet (dashed line) or high-fat LE diet (solid line) and then were quantified for the levels of plasma adiponectin (a), ALT (b), and TNF-α (c), as described in the text. *P < 0.05 for ethanol diet versus control diet (n = 6).
Recombinant adiponectin markedly attenuated ethanol-induced liver injury. We next investigated the effect of adiponectin on alcohol-induced liver injury by infusion of full-length recombinant protein produced from HEK293 cells. Three weeks after being fed with the LE diet, the mice were surgically implanted with an ALZET osmotic pump (DURECT Corp., Cupertino, California, USA), which delivered 30 μg/d adiponectin or physiological saline as control. Delivery of adiponectin at this dosage caused approximately 2.7-fold elevation in the circulating concentration of adiponectin over that of untreated LE mice. The elevated concentration of plasma adiponectin remained constant throughout the course of treatment (Figure 2a). Adiponectin treatment had no obvious effects on food intake, body weight gains, and the levels of blood glucose and insulin (Figure 2, b–e), whereas it could decrease the elevated plasma concentrations of TG and FFA (Figure 3, a and b). Notably, continuous administration of adiponectin for 2 weeks significantly decreased the ratio of liver-to-body weight (Figure 3c), dramatically reduced hepatic lipid content (Figure 3d), and also markedly alleviated alcohol-induced elevation of serum ALT levels (Figure 3e).
Effects of adiponectin treatment on food intake, body weight gains, and circulating levels of glucose and insulin. (a) Serum adiponectin levels in mice fed with LC diet, LE diet, or LE diet plus adiponectin infusion (LE + Ad), as described in the text (n = 5). Serum samples were collected at different times following adiponectin treatment. (b) Food intake in LE diet mice treated with or without adiponectin (n = 6). (c) Average daily body weight gains over 2 weeks of adiponectin treatment (n = 6). (d) Fasting glucose levels over adiponectin treatment period (n = 6). (e) Fasting plasma insulin levels over adiponectin treatment period (n = 6). Note that adiponectin and alcohol treatment also have no obvious effects on plasma levels of insulin and glucose in the fed state (data not shown). *P < 0.001 for adiponectin-treated group versus other two groups.
Adiponectin treatment ameliorates alcohol-induced dyslipidemia and hepatic abnormality in mice. Serum samples were collected after 5 weeks of LC diet, LE diet, or LE + Ad diet in the last 2 weeks. Serum levels of TG (a) and FFA (b), liver-to-body weight ratio (c), hepatic TG contents (d), and plasma ALT levels (e) were determined at necropsy (n = 5). #P < 0.05, ##P < 0.01 for LC-treated mice versus LE-treated mice; *P < 0.05, **P < 0.01 for LE + Ad–treated mice versus LE-treated mice.
Histological evaluation of liver specimens demonstrated massive panlobular microvesicular and macrovesicular steatosis and occasional foci of inflammation in mice fed with LE diet alone (Figure 4). Administration of adiponectin dramatically decreased lipid accumulation to a background level and largely diminished inflammation (as judged by the absence of inflammatory foci under microscopy). Thus, our results demonstrated a protective role of adiponectin in alcohol-induced liver injury in mice.
Effects of adiponectin on alcohol-induced steatosis and inflammation. Liver specimens were taken from mouse livers after 5 weeks of LC diet (a and d), LE diet (b and e), or LE + Ad diet (c and f) for the last 2 weeks and were stained with either red oil O (a–c) or H&E (d–f). The results are representative photomicrographs from six independent experiments.
Administration of adiponectin decreased hepatic expression of TNF-α and its plasma concentrations. Elevated production of TNF-α from Kuppfer cells within the liver tissue is thought to be a key mediator of early alcohol-induced liver injury (5). A recent knockout study suggests that TNF-α and adiponectin may antagonize each other’s actions by each suppressing the other’s expression (19). We thus tested the hypothesis that adiponectin might alleviate alcohol-induced liver injury partly by suppressing alcohol-induced elevation of TNF-α production. Indeed, treatment of LE diet mice with recombinant adiponectin blunted the alcohol-induced increase of circulating TNF-α as well as mRNA production of this cytokine in the liver (Figure 5).
Adiponectin treatment decreases alcohol-induced elevation of hepatic TNF-α expression and its plasma concentrations. (a) Total RNA from livers of mice treated with LC diet, LE diet, or LE + Ad diet was extracted and subjected to Northern blot analysis. (b) The results from a were quantified by PhosphorImaging (n = 5). All RNA levels are expressed relative to untreated LC pair-fed controls, after being normalized against the abundance of 18S RNA. (c) Plasma concentrations of TNF-α as measured at necropsy (n = 5). **P < 0.01 for LE + Ad–treated mice versus mice receiving LE diet alone.
Adiponectin treatment restored alcohol-induced suppression of CPT I activity and enhanced hepatic fatty acid oxidation. Impaired fatty acid oxidation plays a key role in alcoholic steatosis (33). Alcohol has been shown to decrease hepatic fatty acid oxidation in vivo and in vitro. Alcohol metabolism alters the intramitochondrial redox potential, which in turn impairs β oxidation and tricarboxylic acid cycle activity (34). In addition, both long-term and short-term alcohol consumption has been shown to suppress the activity of CPT I, a rate-limiting enzyme involved in the transport of long-chain fatty acids into mitochondrial matrix (35, 36). A truncated globular adiponectin can enhance fatty acid oxidation in muscle (21). We next investigated whether adiponectin-induced depletion of hepatic lipid accumulation was caused partly by enhancing fatty acid oxidation in this tissue. Indeed, consumption of LE diet significantly decreased the rate of hepatic fatty acid oxidation and also reduced CPT I activity (Figure 6). These ethanol-induced alterations were restored following adiponectin treatment.
Effects of adiponectin on alcohol-induced suppression of hepatic CPT I activity (a) and fatty acid oxidation (b). Three groups of animals were treated with LC diet, LE diet, or LE + Ad diet as described in Figure 4. The activity of CPT I was determined by measuring 3H palmitoylcarnitine formed in digitonin-permeabilized hepatocytes. The rates of fatty acid oxidation were analyzed by monitoring nanomoles of [1-14C]palmitate oxidized per gram of liver tissue per minute. #P < 0.05 for LC-treated mice versus LE-treated mice; *P < 0.05 for LE + Ad–treated mice versus LE-treated mice.
Adiponectin treatment decreased the enzyme activities involved in fatty acid synthesis. In addition to the suppression of fatty acid oxidation, enhanced fatty acid synthesis also contributes to alcohol-induced fatty liver, especially at the later stage of alcoholism. Chronic ethanol consumption can increase fatty acid synthesis in humans and rodents by inducing the expression of key enzymes in the lipogenic pathway (37, 38). We next investigated the effects of adiponectin on two key enzymes involved in hepatic lipogenesis, including ACC and FAS. Consumption of the LE diet caused a slight, but not significant increase of hepatic ACC activity (Figure 7a). Compared with the untreated LE diet mice, however, adiponectin caused a marked reduction of this enzyme activity. The activity of hepatic FAS was significantly increased following 5 weeks of LE diet, and the elevated enzyme activity was markedly decreased following adiponectin treatment (Figure 7b). Northern blot analysis revealed that the steady-state mRNA abundance of hepatic ACC was not altered following adiponectin treatment (Figure 7, c and d). Expression of hepatic FAS was significantly elevated following consumption of the LE diet, and adiponectin could suppress the hepatic mRNA expression of this enzyme. In addition, the hepatic expression of CD36, a fatty acid transport protein, was markedly inhibited following adiponectin treatment.
Effects of adiponectin on proteins involved in fatty acid synthesis and uptake. Three groups of animals were treated with LC diet, LE diet, or LE + Ad diet as described in Figure 4. (a) Activity of hepatic ACC expressed as nanomoles of 14C malonyl-CoA produced per milligram of protein per minute. (b) Hepatic FAS activity expressed as nanomoles of 14C-labeled fatty acids produced per milligram of protein per minute. (c) Northern blot analysis of steady-state mRNA levels for ACC, FAS, and CD36. (d) The results from c were quantified by PhosphorImaging (n = 5). All mRNA levels are expressed relative to untreated LC pair-fed controls, after being normalized against the abundance of 18S RNA. #P < 0.05, ##P < 0.01 for LC-treated mice versus LE-treated mice; *P < 0.05, **P < 0.01 for LE + Ad–treated mice versus LE-treated mice.
Adiponectin treatment ameliorates nonalcoholic fatty liver disease in ob/ob mice. NASH, which is often associated with obese individuals and patients with insulin resistance and type 2 diabetes, is an important feature of metabolic syndromes (1, 39). Anti-diabetes drugs might also improve fatty liver disease (40). Recombinant adiponectin has been shown to be effective in increasing insulin sensitivity and in decreasing hyperlipidemia in several animal models (20, 41). Therefore, we investigated the potential effects of adiponectin treatment on fatty liver disease in obese, ob/ob mice, which spontaneously develop hyperinsulinemia, insulin resistance, and steatosis owing to an inherited leptin deficiency (42). Adiponectin expression from adipose tissue is markedly reduced in ob/ob mice (43). Notably, overproduction of TNF-α in the liver tissue has been proposed to play a key role in the pathogenesis of fatty liver disease in this model (5). Fatty liver disease in ob/ob mice was significantly improved by inhibition of hepatic TNF-α overproduction or infusion of anti–TNF-α neutralizing Ab (16, 40).
Consistent with those of LE diet–treated mice (Figure 2a), infusion of adiponectin using osmotic pumps (1.5 mg/kg body weight per day for 2 weeks) elevated plasma adiponectin levels approximately 2.7-fold throughout the course of the treatment. Adiponectin treatments had no obvious effects on body weight gains, food intake, and plasma levels of glucose and insulin, whereas circulating concentration of both TG and FFA were significantly decreased compared with untreated ob/ob mice (Table 1). In line with a recent study (44), infusion of adiponectin into obese ob/ob mice increased glucose tolerance as well as insulin sensitivity (Figure 8, a and b). Notably, adiponectin improved fatty liver disease in this animal model. Following 2 weeks of adiponectin treatment, the liver-to-body weight ratios were significantly reduced (Figure 8c), and this change was associated with the decreases in hepatic lipid contents (Figure 8d) and serum ALT levels (Figure 8e). The increased hepatic production of TNF-α in the obese mice was also significantly suppressed following adiponectin treatment (Figure 8f). Thus, adiponectin is an effective polypeptide for the treatment of obesity-associated nonalcoholic fatty liver disease.
Adiponectin increases insulin sensitivity and ameliorates fatty liver diseases in obese ob/ob mice. Age- and sex-matched lean control mice (lean), ob/ob mice (ob/ob), or ob/ob mice treated with adiponectin (ob/ob + Ad) for 2 weeks were subjected to a glucose-tolerance test (a) and insulin-tolerance test (b). The ratios of liver-to-body weight (c), hepatic TG contents (d), and serum ALT levels (e) were determined at necropsy. Hepatic mRNA expression of TNF-α (f) was analyzed as in Figure 5. The results were from five to seven mice per group. *P < 0.05, **P < 0.01 for adiponectin-treated ob/ob mice versus untreated ob/ob mice.
Effects of adiponectin on body weight gains, food intake, and plasma concentrations of glucose, insulin, TG, and FFA in ob/ob mice
Adiponectin levels negatively correlate with serum ALT concentrations in morbidly obese Chinese individuals. The results above suggest that adiponectin is associated with alcohol- and obesity-induced fatty liver diseases. To explore the clinical relevance of these findings, we next investigated the relationship between plasma levels of adiponectin and of ALT (a marker of liver injury) in 90 morbidly obese Chinese individuals (age 42 ± 9; BMI 40.1 ± 5.2). The rationale for this analysis is that there is a strong association between obesity and NASH. The prevalence of NASH among obese subjects is sixfold higher compared with lean individuals (45). Indeed, after adjustment for age, sex, and BMI, plasma levels of adiponectin inversely correlated with that of ALT (Figure 9), suggesting that hypoadiponectinemia may partly account for high incidence of NASH in obese individuals. Further prospective and cross-sectional studies are needed to ascertain the role of adiponectin in the cause of ASH and NASH in humans.
Relationship between plasma levels of adiponectin and ALT in 90 morbidly obese Chinese individuals (45 male and 45 female). r, Spearman correlation.