Valérie Lebrun

Defective hepatic regeneration after partial hepatectomy in leptin-deficient mice is not rescued by exogenous leptin

Liver regeneration after partial hepatectomy (PH) is impaired in leptin-deficient ob/ob mice. Here, we tested whether exogenous leptin and/or correction of the obese phenotype (by food restriction or long-term leptin administration) would rescue hepatocyte proliferation and whether the hepatic progenitor cell compartment was activated in leptin-deficient ob/ob livers after PH. Because of the high mortality following 70% PH to ob/ob mice, we performed a less extensive (55%) resection. Compared to lean mice, liver regeneration after 55% PH was deeply impaired and delayed in ob/ob mice. Administration of exogenous leptin to ob/ob mice at doses that restored circulating leptin levels during the surgery and postsurgery period or for 3 weeks prior to the surgical procedure did not rescue defective liver regeneration. Moreover, correction of obesity, metabolic syndrome and hepatic steatosis by prolonged administration of leptin or food restriction (with or without leptin replacement at the time of PH) did not improve liver regeneration in ob/ob mice. The hepatic progenitor cell compartment was increased in ob/ob mice. However, after PH, the number of progenitor cells decreased and signs of proliferation were absent from this cell compartment. In this study, we have conclusively shown that neither leptin replacement nor amelioration of the metabolic syndrome, obese phenotype and hepatic steatosis, with or without restitution of normal circulating levels of leptin, was able to restore replicative competence to ob/ob livers after PH. Thus, leptin does not directly signal to liver cells to promote hepatocyte proliferation, and the obese phenotype is not solely responsible for impaired regeneration.

Prevention of steatohepatitis by pioglitazone: implication of adiponectin-dependent inhibition of SREBP-1c and inflammation.

BACKGROUND/AIMS Peroxisome proliferator-activated receptor gamma (PPARgamma) agonist drugs, like pioglitazone (PGZ), are proposed as treatments for steatohepatitis. Their mechanisms of action remain ill-clarified. METHODS To test the hypothesis that PGZ improves steatohepatitis through adiponectin-dependent stimulation of AMPK and/or PPARalpha, mice lacking adiponectin (Adipo(-/-)) or the AMPKalpha1 catalytic subunit (AMPKalpha1(-/-)) or wild-type (Wt) mice were fed the methionine and choline deficient (MCD) diet, supplemented or not with PGZ. RESULTS In Wt mice, PGZ increased circulating levels of adiponectin and reduced the severity of MCD-induced steatohepatitis but there was no evidence of activation of AMPK or PPARalpha and their downstream targets. By contrast, PGZ completely repressed nuclear translocation of SREBP-1c, a key transcription factor for de novo lipogenesis. This effect was lacking in Adipo(-/-) mice in which PGZ failed to prevent steatohepatitis. Surprisingly, AMPKalpha1(-/-) mice were resistant to MCD-induced steatohepatitis, a status also associated with repression of SREBP-1c. CONCLUSIONS The preventive effect of PGZ on MCD-induced steatohepatitis depends on adiponectin upregulation but apparently does not involve AMPK or PPARalpha activation. The inhibition of SREBP-1c and dependent repression of lipogenesis are likely to participate in this effect. The mechanisms by which PGZ and adiponectin control SREBP-1c and inflammation remain to be elucidated.

The IGF2 mRNA binding protein p62/IGF2BP2-2 induces fatty acid elongation as a critical feature of steatosis

Liver-specific overexpression of the insulin-like growth factor 2 (IGF2) mRNA binding protein p62/IGF2BP2-2 induces a fatty liver, which highly expresses IGF2. Because IGF2 expression is elevated in patients with steatohepatitis, the aim of our study was to elucidate the role and interconnection of p62 and IGF2 in lipid metabolism. Expression of p62 and IGF2 highly correlated in human liver disease. p62 induced an elevated ratio of C18:C16 and increased fatty acid elongase 6 (ELOVL6) protein, the enzyme catalyzing the elongation of C16 to C18 fatty acids and promoting nonalcoholic steatohepatitis in mice and humans. The p62 overexpression induced the activation of the ELOVL6 transcriptional activator sterol regulatory element binding transcription factor 1 (SREBF1). Recombinant IGF2 induced the nuclear translocation of SREBF1 and a neutralizing IGF2 antibody reduced ELOVL6 and mature SREBF1 protein levels. Concordantly, p62 and IGF2 correlated with ELOVL6 in human livers. Decreased palmitoyl-CoA levels, as found in p62 transgenic livers, can explain the lipogenic action of ELOVL6. Accordingly, p62 represents an inducer of hepatic C18 fatty acid production via a SREBF1-dependent induction of ELOVL6. These findings underline the detrimental role of p62 in liver disease.

Impact of PPAR-α induction on glucose homoeostasis in alcohol-fed mice.

Alcohol consumption is a major cause of liver disease. It also associates with increased cardiovascular risk and Type 2 diabetes. ALD (alcoholic liver disease) and NAFLD (non-alcoholic fatty liver disease) share pathological features, pathogenic mechanisms and pattern of disease progression. In NAFLD, steatosis, lipotoxicity and liver inflammation participate to hepatic insulin resistance. The aim of the present study was to verify the effect of alcohol on hepatic insulin sensitivity and to evaluate the role of alcohol-induced steatosis and inflammation on glucose homoeostasis. C57BL/6J mice were fed for 20 days a modified Lieber-DeCarli diet in which the alcohol concentration was gradually increased up to 35% of daily caloric intake. OH (alcohol liquid diet)-fed mice had liver steatosis and inflammatory infiltration. In addition, these mice developed insulin resistance in the liver, but not in muscles, as demonstrated by euglycaemic-hyperinsulinaemic clamp and analysis of the insulin signalling cascade. Treatment with the PPAR-α (peroxisome-proliferator-activated receptor-α) agonist Wy14,643 protected against OH-induced steatosis and KC (Kupffer cell) activation and almost abolished OH-induced insulin resistance. As KC activation may modulate insulin sensitivity, we repeated the clamp studies in mice depleted in KC to decipher the role of macrophages. Depletion of KC using liposomes-encapsuled clodronate in OH-fed mice failed both to improve hepatic steatosis and to restore insulin sensitivity as assessed by clamp. Our study shows that chronic alcohol consumption induces steatosis, KC activation and hepatic insulin resistance in mice. PPAR-α agonist treatment that prevents steatosis and dampens hepatic inflammation also prevents alcohol-induced hepatic insulin resistance. However, KC depletion has little impact on OH-induced metabolic disturbances.

Molecular mechanisms of apoptosis in the liver of rats after portal branch ligation with and without retrorsine

The mechanisms accounting for the atrophy of the portal blood-deprived liver lobes after portal branch ligation (PBL) are still unclear. The first aim of this study was to confirm the role of apoptosis in this process and to determine which apoptotic pathways are involved. The second aim of the study was to evaluate the effect of blocking compensatory hyperplasia of the nonligated lobes with retrorsine on the mechanisms of apoptosis in the ligated lobes. Mitochondrial Bax, Bcl-2 and Bcl-XL, cytosolic cytochrome c, caspase-3, -8 and -9 activities and TNF-α levels were assessed in the liver of rats before and at various time points, ranging from 30 min to 7 days, after PBL. Caspase activities were also measured in rats pretreated with retrorsine. Both the mitochondrial and the death receptor-mediated pathways are activated in the ligated liver lobes after portal branch ligation. Caspase activation is inhibited by retrorsine pretreatment, resulting in fewer apoptotic bodies. Apoptosis accounts for the atrophy of the ligated lobes after PBL. It is inhibited by retrorsine, suggesting an attempt to reduce the loss of liver mass when hyperplasia of the nonligated lobes is impaired.

Inhibition of early preneoplastic events in the rat liver by the somatostatin analog lanreotide

Hepatocellular carcinoma (HCC) is the third most common cause of cancer‐related death, and its incidence is increasing worldwide. Due to the known risk factors (mainly hepatitis B and C viruses), we believe there is a rationale for a chemopreventive approach to treat HCC. Here, based on described in vitro data, we evaluated the preventive effects of lanreotide, a somatostatin analog, on the induction of early carcinogenic events. We monitored preneoplastic foci induced by a two‐stage initiation/promotion model of hepatocarcinogenesis in male Wistar rats, using diethylnitrosamine and 2‐acetylaminofluorene. Lanreotide was given starting the day after the first diethylnitrosamine injection. By quantitative morphometry, we showed that lanreotide significantly decreases the size of induced preneoplastic foci. Analysis of proliferation and apoptosis assessed by immunohistochemistry, showed decreased proliferation and increased cell death in rats treated with lanreotide. As these events were associated with a significant decreased expression of the cell cycle regulator cyclin D1 and an increased expression of the cyclin‐dependent kinase inhibitor p27kip1 compared to the non‐treated group, it is tempting to speculate that these factors are involved in the favorable effect of lanreotide. In conclusion, lanreotide significantly decreases early carcinogenic transformation in a two‐step rat model. As lanreotide has a low toxicity profile, we believe it would be interesting to evaluate its effect in chemoprevention of HCC. (Cancer Sci 2007; 98: 1831–1839)

P0950 : Impact of kupffer cells on high fat induced insulin resistance and liver fetuin-A expression

Backgroun and aims: Hepatokines (liver secreted proteins with possible distant action) are emerging potential players in insulin resistance in type 2 diabetic patients. Here, we explore the effect of a high fat diet on the expression of fetuin-A, one of those candidate liver proteins, and its relation with liver macrophage (Kupffer cell) activation. Methods: Male mice of 5 weeks of age were fed a normal diet (ND) or a high fat diet (HFD) for 3 days, known to initiate steatosis and insulin resistance. A preventive Kupffer cell (KC) depletion was obtained by intravenous injection of clodronate loaded liposomes and compared with PBS liposomes. The mRNA and protein expression of fetuin-A was evaluated by RT-PCR, Western-blot and immunofluorescence (IF) on different insulin-sensitive tissues (liver, adipose tissue and muscle). Results: Short term HFD induced steatosis, KC activation and insulin resistance together with a significant increased expression of liver fetuin-A mRNA (1.5 fold, p<0.01). However, liver fetuin-A protein expression remained unchanged under short term HFD. This increase in fetuin-A under high fat diet was not evidenced in the peripheral insulin sensitive tissues (skeletal muscle and adipose tissue) whether at the mRNA or at the protein level. Kupffer cell depletion in this setting did not reduce hepatic steatosis but significantly ameliorated insulin sensitivity proved by clamp studies. This amelioration in insulin sensitivity in KC-depleted mice was associated with a significant decrease in fetuin-A mRNA expression (0.7 fold, p<0.01) compared to animals with KC. On immunofluorescence, fetuin-A was mostly expressed in centrilobular hepatocytes. Interestingly, while selectively depleting liver macrophages without affecting adipose tissue macrophage infiltration, intravenous clodronate injection was associated with a significant reduction in epididymal adipose tissue expansion compared to PBS injection (1.1% of body weight versus 1.6% of body weight, p<0.001). Conclusion: This study demonstrates liver fetuin-A overexpression at the initiation of HFD feeding, concurrent with hepatic steatosis and insulin resistance. Targeting KC in this setting improved insulin sensitivity and was associated with a decreased adiposity and a reduced liver fetuin-A expression suggesting that fetuin-A acts as an hepatokine with pro-adiposity and pro-insulin resistance effects.