Endocannabinoids and their role in fatty liver disease.

The endocannabinoid system comprises receptors, CB1 and CB2, their endogenous lipidic ligands and machinery dedicated to endocannabinoid synthesis and degradation. An overactive endocannabinoid system appears to contribute to the pathogenesis of several diseases, including liver diseases. With the increasing incidence of non-alcoholic fatty liver disease (NAFLD) in parallel with the obesity epidemic, the development of effective therapies is gaining considerable interest. Several recent experimental lines of evidence identify CB receptors as potential novel therapeutic targets in the management of NAFLD. Endogenous activation of peripheral CB1 receptors is a key mediator of insulin resistance and enhances liver lipogenesis in experimental models of NAFLD. Moreover, we have shown that adipose tissue CB2 receptors are markedly upregulated and promote fat inflammation, thereby contributing to insulin resistance and liver steatosis. Data from our group also indicate that tonic activation of CB1 receptors is responsible for progression of liver fibrosis, whereas CB2 receptors display anti-fibrogenic properties. The clinical relevance of these findings is supported by studies in patients with chronic hepatitis C indicating that daily cannabis use is an independent predictor of both fibrosis and steatosis severity. Moreover, preliminary data derived from clinical trials strongly suggest that selective CB1 antagonism improves insulin resistance and reduces liver fat. Tempering these promises, the first generation of CB1 antagonists raised concern due to an alarming rate of mood disorders and the development program of these molecules was suspended. Current research efforts are therefore focused on developing formulations of CB1 antagonists that do not enter the central nervous system, and preliminary experimental data obtained with such molecules are encouraging.

[Liver fibrosis: from pathophysiology to therapeutic openings]. / Fibrose hépatique: de la physiopathologie aux implications thérapeutiques.

Understanding of liver fibrosis pathogenesis has undergone tremendous advances over the past twenty years. In this respect, demonstration of the reversibility of fibrosis was a major turnpoint. The panel of therapeutic targets is continuously expanding. Clinical development has however remained limited, heretofore, but should rapidly progress owing to the availability of accurate non-invasive methods for assessment of fibrosis, to improvement in the selection patients included in therapeutic trials, and to the development of cell specific targeting devices for agents at risk of adverse effects.

Cellular targeting of the apoptosis-inducing compound gliotoxin to fibrotic rat livers.

Liver fibrosis is associated with proliferation of hepatic stellate cells (HSCs) and their transformation into myofibroblastic cells that synthesize scar tissue. Several studies indicate that induction of apoptosis in myofibroblastic cells may prevent fibrogenesis. Gliotoxin (GTX) was found to induce apoptosis of hepatic cells and caused regression of liver fibrosis. However, the use of apoptosis-inducing drugs may be limited due to lack of cell specificity, with a risk of severe adverse effects. In previous studies, we found that mannose-6-phosphate-modified human serum albumin (M6P-HSA) selectively accumulated in liver fibrogenic cells. The aim of this study therefore was to couple GTX to M6P-HSA and test its pharmacological effects in vitro and in rats with liver fibrosis. The conjugate GTX-M6P-HSA bound specifically to HSCs and reduced their viability. Apoptosis was induced in cultures of human hepatic myofibroblasts (hMFs) and in liver slices obtained from rats with liver fibrosis. In vivo treatment with GTX or GTX-M6P-HSA in bile duct ligated rats revealed a significant decrease in alpha-smooth muscle actin mRNA levels and a reduced staining for this HSC marker in fibrotic livers. In addition, although GTX also affected hepatocytes, GTX-M6P-HSA did not significantly affect other liver cells. In conclusion, we developed an HSC-specific compound that induced apoptosis in human hMFs, rat HSCs, and in fibrotic liver slices. In vivo, both GTX and GTX-M6P-HSA attenuated the number of activated HSCs, but GTX also affected hepatocytes. This study shows that cell-selective delivery of the apoptosis-inducing agent GTX is feasible in fibrotic livers.

CB2 receptors as new therapeutic targets for liver diseases.

Cannabinoid type-1 (CB1) and type-2 (CB2) receptors belong to the family of G protein-coupled receptors and mediate biological effects of phyto-derived and endogenous cannabinoids. Whereas functions of CB1 receptor have been extensively studied, the CB2 receptor has emerged over the last few years as a critical player in regulation of inflammation, pain, atherosclerosis and osteoporosis. Therefore, although still at a preclinical stage, the development of selective CB2 molecules has gained of interest as new targets in drug discovery. Recent data have unravelled a key role of CB2 receptors during chronic and acute liver injury, including fibrogenesis associated to chronic liver diseases, ischaemia-reperfusion-induced liver injury, and hepatic encephalopathy associated to acute liver failure. This review summarizes the latest advances on the recently identified role of CB2 receptors in the pathophysiology of liver diseases.

Cannabinoid receptors as novel therapeutic targets for the management of non-alcoholic steatohepatitis.

Prevalence of non-alcoholic steatohepatitis (NASH) rises steadily in Western countries with the obesity epidemic. NASH is associated with activation of liver fibrogenesis and predisposes to cirrhosis and associated morbi-mortality. The cannabinoid system is increasingly emerging as a crucial mediator of acute and chronic liver injury. Recent experimental and clinical data indicate that peripheral activation of cannabinoid CB1 receptors promotes insulin resistance and liver steatogenesis, two key steps in the pathogenesis of non-alcoholic fatty liver disease. Moreover, CB1 receptors enhance progression of liver fibrogenesis. These findings provide a strong rationale for the use of CB1 antagonists in the management of NASH.

Chronic hypocalcemia of vitamin D deficiency leads to lower intracellular calcium concentrations in rat hepatocytes.

Several lines of evidence indicate that calcium deficiency is associated with cellular defects in many tissues and organs. Owing to the large in vivo gradient between ionized extra- and intracellular Ca2+ concentrations ([Ca2+]i), it is generally recognized that the prevailing circulating Ca2+ does not significantly affect resting cytosolic Ca2+. To probe the consequences of hypocalcemia on [Ca2+]i, a model of chronic hypocalcemia secondary to vitamin D (D) deficiency was used. Hepatocytes were isolated from livers of hypocalcemic D-deficient, of normocalcemic D3-repleted, or of normal control rats presenting serum Ca2+ of 0.78 +/- 0.02, 1.24 +/- 0.03, or 1.25 +/- 0.01 mM, respectively (P < 0.0001). [Ca2+]i was measured in cell couplets using the fluorescent probe Fura-2. Hepatocytes of normocalcemic D3-repleted and of normal controls exhibited similar [Ca2+]i of 227 +/- 10 and 242 +/- 9 nM, respectively (NS), whereas those of hypocalcemic rats had significantly lower resting [Ca2+]i (172 +/- 10 nM; P < 0.0003). Stimulation of hepatocytes with the alpha 1-adrenoreceptor agonist phenylephrine illicited increases in cytosolic Ca2+ leading to similar [Ca2+]i and phosphorylase a (a Ca(2+)-dependent enzyme) activity in all groups but in contrast to normocalcemia, low extracellular Ca2+ was often accompanied by a rapid decay in the sustained phase of the [Ca2+]i response. When stimulated with the powerful hepatic mitogen epidermal growth factor (EGF), hepatocytes isolated from hypocalcemic rat livers responded with a blunted maximal [Ca2+]i of 237.6 +/- 18.7 compared with 605.2 +/- 89.9 nM (P < 0.0001) for their normal counterparts, while the EGF-mediated DNA synthesis response was reduced by 50% by the hypocalcemic condition (P < 0.03). Further studies on the possible mechanisms involved in the perturbed [Ca2+]i homeostasis associated with chronic hypocalcemia revealed the presence of an unchanged plasma membrane Ca2+ ATPase but of a significant decrease in agonist-stimulated Ca2+ entry as indicated using Mn2+ as surrogate ion (P < 0.03). Our data, thus indicate that, in rat hepatocytes, the in vivo calcium status significantly affects resting [Ca2+]i, and from this we raise the hypothesis that this lower than normal [Ca2+]i may be linked, in calcium disorders, to inappropriate cell responses mediated through the calcium signaling pathway as illustrated by the response to phenylephrine and EGF.

Endothelin action in rat liver. Receptors, free Ca2+ oscillations, and activation of glycogenolysis.

High affinity binding sites for endothelin (ET) were identified on rat liver plasma membranes. Binding of 125I-ET-1 with its site was specific, saturable, and time dependent (kobs = 0.019 +/- 0.001 min-1), but dissociation of receptor-bound ligand was minimal. A single class of high affinity binding sites for 125I-ET-1 was identified with an apparent Kd of 32.4 +/- 9.8 pM and a Bmax of 1084 +/- 118 fmol/mg protein. ET-3 and big-ET-1 (1-38) (human) inhibited 125I-ET-1 binding with IC50 values of 1.85 +/- 1.03 nM and 43 +/- 6 nM, respectively. Aequorin measurements of cytosolic free Ca2+ in single, isolated rat hepatocytes showed that ET-1 at subnanomolar concentrations induced a series of repetitive, sustained Ca2+ transients. ET-1 had no effect on cAMP production. Finally, ET-1 caused a rapid and sustained stimulation of glycogenolysis in rat hepatocytes. A 1.8-fold maximal increase in glycogen phosphorylase alpha was observed at 1 pM ET-1, with an EC50 of 0.03 pM. Stimulation of the enzyme was specific for ET-1 since the order of potency of related peptides was similar to that in binding experiments (ET-1 greater than ET-3 greater than big ET-1). These data constitute the first demonstration of the presence of ET-1 binding sites in liver which is associated with a rise in cytosolic free Ca2+ and a potent glycogenolytic effect. We conclude that ET-1 behaves as a typical Ca2+ mobilizing hormone in liver.

Growth inhibitory properties of endothelin-1 in human hepatic myofibroblastic Ito cells. An endothelin B receptor-mediated pathway.

Ito cells play a pivotal role in the development of liver fibrosis associated with chronic liver diseases. During this process, Ito cells acquire myofibroblastic features, proliferate, and synthesize fibrosis components. Considering the reported mitogenic properties of endothelin-1 (ET-1), we investigated its effects on the proliferation of human Ito cells in their myofibroblastic phenotype. Both ET receptor A (ETA: 20%) and ET receptor B (ETB: 80%) binding sites were identified, using a selective ETA antagonist, BQ 123, and a selective ETB agonist, sarafotoxin S6C (SRTX-C). ET-1 did not stimulate proliferation of myofibroblastic Ito cells. In contrast, ET-1 inhibited by 60% DNA synthesis and proliferation of cells stimulated with either human serum or platelet-derived growth factor -BB (PDGF-BB). PD 142893, a nonselective ETA/ETB antagonist totally blunted this effect. SRTX-C was as potent as ET-1, while BQ 123 did not affect ET-1-induced growth inhibition. Analysis of the intermediate steps leading to growth-inhibition by ET-1 revealed that activation of mitogen-activated protein kinase by serum or PDGF-BB was decreased by 50% in the presence of SRTX-C. In serum-stimulated cells, SRTX-C reduced c-jun mRNA expression by 50% whereas c-fos or krox 24 mRNA expression were not affected. We conclude that ET-1 binding to ETB receptors causes a potent growth inhibition of human myofibroblastic Ito cells, which suggests that this peptide could play a key role in the negative control of liver fibrogenesis. Our results also point out that, in addition to its well known promitogenic effects, ET-1 may also exert negative control of growth on specific cells.

Growth inhibitory properties of endothelin-1 in activated human hepatic stellate cells: a cyclic adenosine monophosphate-mediated pathway. Inhibition of both extracellular signal-regulated kinase and c-Jun kinase and upregulation of endothelin B receptors.

During chronic liver diseases, hepatic stellate cells (HSC) acquire an activated myofibroblast-like phenotype, proliferate, and synthetize fibrosis components. We have shown that endothelin-1 (ET-1) inhibits the proliferation of activated human HSC via endothelin B (ETB) receptors. We now investigate the transduction pathway involved in the growth inhibitory effect of ET-1 in activated HSC. Endothelin-1 and the ETB receptor agonist, sarafotoxin-S6C, increased synthesis of PGI2 and PGE2, leading to elevation of cAMP. The cyclooxygenase inhibitor ibuprofen and the adenylyl cyclase inhibitor SQ22536 both blunted the growth inhibitory effect of ET-1. Analysis of early steps associated with growth inhibition indicated that: (a) similar to ET-1, forskolin decreased c-jun mRNA induction without affecting c-fos and krox 24 mRNA expression; (b) ET-1, sarafotoxin-S6C, as well as forskolin, reduced activation of both c-Jun kinase and extracellular signal-regulated kinase. Finally, forskolin, PGI2, and PGE2 raised by fivefold the number of ET binding sites after 6 h, and increased the proportion of ETB receptors from 50% in control cells to 80% in treated cells. In conclusion, ET-1 inhibits proliferation of activated HSC via ETB receptors, through a prostaglandin/cAMP pathway that leads to inhibition of both extracellular signal-regulated kinase and c-Jun kinase activities. Upregulation of ETB receptors by prostaglandin/cAMP raises the possibility of a positive feedback loop that would amplify the growth inhibitory response. These results suggest that ET-1 and agents that increase cAMP might be of interest to limit proliferation of activated HSC during chronic liver diseases.

The liver plasma membrane Ca2+ pump: hormonal sensitivity.

The liver plasma membrane Ca2+ pump is supposed to extrude cytosolic calcium out of the cell. This system has now been well defined on the basis of its plasma membrane origin, its high affinity Ca2+ -stimulated ATPase activity, its Ca2+ transport activity, its phosphorylated intermediate. The liver calcium pump appears to be a target of hormonal action since it has been shown that glucagon and calcium mobilizing hormones namely alpha 1-adrenergic agonists, vasopressin, angiotensin II inhibit this system. The present review details the mechanism of calcium pump inhibition by glucagon and points out its difference from the inhibition process induced by calcium mobilizing hormones. We conclude that the inhibitory action of the Ca2+ mobilizing hormones and glucagon on the liver plasma membrane Ca2+ pump might play a key role in the actions of these hormones by prolonging the elevation in cytosolic free Ca2+.

The endocannabinoid system as a key mediator during liver diseases: new insights and therapeutic openings.

Chronic liver diseases represent a major health problem due to cirrhosis and its complications. During the last decade, endocannabinoids and their receptors have emerged as major regulators of several pathophysiological aspects associated with chronic liver disease progression. Hence, hepatic cannabinoid receptor 2 (CB(2)) receptors display beneficial effects on alcoholic fatty liver, hepatic inflammation, liver injury, regeneration and fibrosis. Cannabinoid receptor 1 (CB(1)) receptors have been implicated in the pathogenesis of several lesions such as alcoholic and metabolic steatosis, liver fibrogenesis, or circulatory failure associated with cirrhosis. Although the development of CB(1) antagonists has recently been suspended due to the high incidence of central side effects, preliminary preclinical data obtained with peripherally restricted CB(1) antagonists give real hopes in the development of active CB(1) molecules devoid of central adverse effects. CB(2) -selective molecules may also offer novel perspectives for the treatment of liver diseases, and their clinical development is clearly awaited. Whether combined treatment with a peripherally restricted CB(1) antagonist and a CB(2) agonist might result in an increased therapeutic potential will warrant further investigation.

Endocannabinoids and liver disease. I. Endocannabinoids and their receptors in the liver.

Cannabinoid receptors (CB1 and CB2) and their endogenous ligands (endocannabinoids) have recently emerged as novel mediators of liver diseases. Endogenous activation of CB1 receptors promotes nonalcoholic fatty liver disease (NAFLD) and progression of liver fibrosis associated with chronic liver injury; in addition, CB1 receptors contribute to the pathogenesis of portal hypertension and cirrhotic cardiomyopathy. CB2 receptor-dependent effects are also increasingly characterized, including antifibrogenic effects and regulation of liver inflammation during ischemia-reperfusion and NAFLD. It is likely that the next few years will allow us to delineate whether molecules targeting CB1 and CB2 receptors are useful therapeutic agents for the treatment of chronic liver diseases.

[The endocannabinoid system as a novel target for the treatment of liver fibrosis]. / Le système endocannabinoïde, une nouvelle cible pour le traitement de la fibrose hépatique.

The cannabinoid system comprises specific G protein-coupled receptors (CB1 and CB2), exogenous (marijuana-derived cannabinoids) and endogenous (endocannabinoids) ligands, and a machinery dedicated to endocannabinoid synthesis and degradation. Studies over two decades have extensively documented the crucial role of the cannabinoid system in the regulation of a variety of pathophysiological conditions. However, its role in liver pathology has only been recently unravelled, probably given the low expression of CB1 and CB2 in the normal liver. We have recently demonstrated that CB1 and CB2 receptors display opposite effects in the regulation of liver fibrogenesis during chronic liver injury. Indeed, both receptors are up-regulated in the liver of cirrhotic patients, and expressed in liver fibrogenic cells. Moreover, CB1 receptors are profibrogenic and accordingly, the CB1 antagonist rimonabant reduces fibrosis progression in three experimental models. In keeping with these results, daily cannabis smoking is a risk factor for fibrosis progression in patients with chronic hepatitis C. In contrast, CB2 display antifibrogenic effects, by a mechanism involving reduction of liver fibrogenic cell accumulation. These results may offer new perspectives for the treatment of liver fibrosis, combining CB2 agonist and CB1 antagonist therapy.