Cynthia R.L. Webster, DVM, DACVIM
By virtue of what they do, hepatocytes are uniquely in harm's way. Their role in the detoxification and clearance of endogenous metabolites and xenobiotics exposes them to potential toxic intermediates. Since they receive the majority of their blood supply from the poorly oxygenated portal system which drains the gastrointestinal tract, they are highly susceptible to ischemic injury and at risk for exposure to potentially toxic substances absorbed across the GI mucosa. In addition, the liver has a large population of macrophages (Kupffer cells) that are poised for activation and release of toxic cytokines. Hepatocytes thus have developed a secure armor to protect themselves from harm which includes enzymatic (catalase, superoxide dismutase and glutathione peroxidase and transfersase) and nonenzymatic defense (glutathione, vitamin E) mechanisms. They also respond to toxic signals by initiating intracellular pro-survival biochemical pathways. These pathways controlled by hormones such as glucagon and growth factors such as hepatocyte growth factor (HGF) work through the modulation of survival kinases.
Mechanisms of Hepatocyte Cell Death
Injured hepatocytes die by either apoptosis or necrosis.1 During necrosis an overwhelming cellular stress results in the depletion of ATP and subsequent accumulation of Ca+2 and reactive oxygen species (ROS) that leads to activation of calpain proteases and lipid peroxidation followed by widespread membrane disruption. Ultimately the hepatocyte rupture which generates an intense inflammatory response. Apoptosis is a genetically controlled pathway to cell death that relies on the activation of a cascade of cellular proteases called caspases. Caspase activation occurs by either a death receptor or a mitochondrial mediated pathway. In the former, binding of the death receptor ligands, tumor necrosis factor-(TNF) and Fas results in receptor oligomerization and activation. This leads to recruitment of the adapter molecule, FADD (Fas Associated protein with Death Domain), to the death domain of the receptor. Procaspase-8 binds to FADD and the death inducing signal complex (DISC) is formed. Procaspase-8 is activated by auto-cleavage within the DISC and in Type 1 cells such as immunocytes directly cleaves caspase 3. Caspase 3 is the final effector caspase and once activated apoptosis proceeds. In Type II cells, like hepatocytes, only a small amount of cleaved caspase 8 is generated and goes on to cleave a cytoplasmic member of the Bcl-2 like family of proteins, BID. Caspase-8 cleavage of BID leads to mitochondrial translocation and/or activation of the pro-apoptotic Bcl-2 proteins, Bax and/or Bak, subsequent disruption of the outer mitochondrial membrane and release of cytochrome C into the cytosol. Cytochrome C stimulates the assembly of the apoptosome, a complex of Apaf-1 (apoptotic protease activating factor 1), procaspase-9, and ATP. Caspase-9 is cleaved within the apoptosome and goes on to cleave and activate caspase-3. In the mitochondrial pathway, the trigger for apoptosis is a direct mitochondrial insult leading to Bax/Bak activation and subsequent mitochondrial membrane permeabilization leading to cytochrome C release. Morphologically apoptosis is marked by cytoplasmic condensation and nuclear chromatin fragmentation and cleavage with preservation of membrane integrity and clearance of the resultant apoptotic bodies by phagocytic cells.
Cellular injury can also induce autophagy which can be a form of cell death or a temporary bridge to cell survival.2 Autophagy is marked morphologically by the formation of autophagic vesicles around damaged mitochondria and proteins. These double-membrane vacuoles fuse with lysosomes eventually resulting in the degradation of the contents. Persistent autophagy, which depletes the cellof organelles and critical proteins, leads to a caspase-independentform of cell death. Autophagy is reversible and can be activated in a more transient fashion (particularly in cancercells) and promotes survival by serving as an intracellular mechanism bywhich cells dispose of damaged organelles and proteins and recycle macromolecules to maintain bioenergetics.
Survival Signaling Pathways
Tyrosine Kinase Receptors
Growth factors such HGF, epidermal growth factor and insulin, promote survival in hepatocytes. When these growth factors bind to their plasma membrane receptors the receptor becomes auto-phosphorylated and the receptors' intrinsic tyrosine kinase activity is activated. This tyrosine kinase activity is coupled to activation of several downstream signal transduction pathways. The pathway most consistently implicated in growth factor survival signaling is the lipid kinase, phosphoinositide-3-kinase (PI3K). The downstream mediators of PI3K survival signaling include the serine/threonine protein kinases, Akt and glycogen synthase kinase (GSK-3β).3 These kinases promote survival by interfering with a family of apoptosis regulators called the Bcl-2 like family of proteins. These proteins are regulators of mitochondrial integrity and can be pro-apoptotic (see Bid, Bax and Bak above) or antiapoptotic (Bcl-2, Bcl-xL, Mcl-1). Akt can phosphorylate and inactivate; 1) Bad, an pro-apoptotic Bcl-2 protein, 2) the transcription factors, FOXO and p53, both of which promote production of pro-apoptotic Bcl-2 like proteins, and 3) GSK-3β and by doing so increase stabilize the anti-apoptotic protein, Mcl-1.
The therapeutic potential of HGF has been demonstrated in vivo4. When bile duct ligated mice were given recombinant HGF it ameliorated hepatobiliary injury. Recent studies have suggested that HGF signaling may be perturbed in canine inflammatory and vascular disease.5,6
G Protein Coupled Receptors
Many G-protein coupled receptors, such as glucagon, transduce intracellular signals by increasing cAMP levels. Cyclic cAMP is cytoprotective in a variety of epithelial cells. Our lab has shown that increasing cAMP levels with cell permeable, phosphodiesterase resistant cAMP analogues protects against bile acid, Fas and TNF mediated hepatocyte apoptosis.7 Cytoprotection does not proceed through classical protein kinase A activation, but instead is mediated by cAMP activation of cAMP-gaunine exchange factors (cAMP-GEF) (Figure 1). cAMP-GEF's are coupled to activation of the small GTPase, Rap.8 cAMP-GEF survival signaling in hepatocytes involves sequential activation of the nonreceptor tyrosine kinase, Src, and PI3K/Akt. This survival pathway prevents bile acid induced activation of caspase 3 and the mitochondrial release of cytochrome C but does not alter the translocation of BAX to the mitochondrial membrane suggesting that cAMP's protective effect is at the level of the mitochondria. Since cAMP is an important second messenger in hepatic stress hormone signaling, increases in intracellular cAMP may represent a general means whereby hepatocytes up-regulate survival mechanisms during times of metabolic stress.
Activation of the Transcription Factor, NF-κβ
NF-κβ, a transcription factor that regulates the expression of a number of pro-inflammatory and anti-apoptotic genes, plays an important role in hepatobiliary pathophysiology.9 A complete knock-out of NK-κβ in mice is embryonically lethal due to hepatocyte apoptosis. NK-κβ activation is particularly important in protecting hepatocytes from injury associated with TNF. TNF is an important mediator of hepatocyte injury as it is readily released from Kupffer cells activated by toxins such as lipopolysaccharide (LPS) that are absorbed from the GI tract. NF-κβ's protective effects include increased expression of anti-apoptotic proteins such as Bcl-xL and FLIP (an inhibitor of the death receptor DISC) and inhibition of activation of pro-apoptotic kinases such as c-Jun terminal kinase (JNK). The role of NK-κβ in hepatocyte survival, however, is complex due to its dual role in inflammation and apoptosis. Strong activation, especially in the face of ischemia-reperfusion worsens hepatocyte injury due to the activation of self-perpetuating inflammatory cascades.
Glutathione (GSH) is the major anti-oxidant in the liver.10,11 It is tripeptide synthesized in the hepatocytes by sequential enzymatic reactions that join glutamine, cysteine and glycine. The rate limiting step in the formation of GSH is the bioavailability of cysteine. Cysteine is derived from the transformation of methionine into cysteine via the transsulfuration pathway (Figure 2) and uptake of dietary cysteine by hepatocytes. The anti-oxidant activity of GSH is associated with: 1) GSH conjugation reactions catalyzed by GSH transferases, and 2) redox cycling (Figure 3). In the latter, GSH oxidation to GSSG is catalyzed by GSH peroxidase and is coupled to conversion of H2O2 to H2O. The generated GSSG is either reduced back to GSH (catalyzed by GSH reductase) or is rapidly effluxed from the cell. Ninety percent of the hepatic GSH is in the cytosol and 10% is mitochondria. Mitochondrial GSH comes from transport of cytosolic GSH into the mitochondrial matrix. Since mitochondria are major generators of ROS, depletion of mitochondrial GSH is particularly detrimental to hepatic function. Hepatocytes can become GSH depleted by an increased rate of utilization due to increased oxidative stress or by decreased synthesis due to decreased availability of cysteine secondary to malnutrition or defective transsulfuration. Since the half life of GSH is only 2-3 hrs, anorexia alone can cause a significant fall in GSH (50% in 48 hrs in rats). In chronic hepatic disorders, SAMe can become a conditionally essential nutrient due to loss of methyladenotransferase (MAT) activity, an enzyme that catalyzes the conversion of methionine to SAMe (Figure 2). Hepatic glutathione levels can be increased by supplementation with n-acetyl cysteine or SAMe. In experimental animals, GSH depletion increases hepatocyte sensitivity to ROS and potentiates TNF and Fas apoptosis.
Hepatocytes require contact with extracellular matrix (ECM) to inhibit detachment-induced apoptosis.12 The attachment of hepatocytes to ECM is mediated by integrins. Integrins link the ECM to the hepatocyte intracellular actin cytoskeleton and can stimulate cellular signaling cascades. Signaling cascades involved in ECM mediated survival include activation of the nonreceptor tyrosine kinase, focal adhesion kinase (FAK), and inhibition of p53 mediated apoptosis. Recent studies in our laboratory have shown that bile acid induced apoptosis is associated with inactivation of FAK signaling and that pretreatment with agents that increase cAMP-GEF signaling can prevent this interruption and rescue hepatocytes from apoptosis.13
N-acetyl cysteine (NAC), a precursor of l-cysteine, can replenish hepatic GSH stores.14,15 NAC is a proven antidote for acetaminophen hepatotoxicity and can protect against hepatic injury due to azathioprine, LPS, viruses, ischemia-reperfusion and bile duct ligation. NAC is also cytoprotective in non-hepatic diseases associated with enhanced oxidative stress such as chronic obstructive pulmonary disease, acute pancreatitis, and renal drug toxicity. Besides antagonizing the effect of intracellular ROS generation, NAC can increase the bioavailability of nitric oxide and decrease the expression of leukocyte adhesion molecules. Some of these effects may be mediated by inhibition of NK-κβ activation and inhibition of toll like receptor signaling in macrophages. The overall effect of NAC is to reverse endothelial cell dysfunction and promote tissue oxygenation. Currently NAC is undergoing clinical trials to treat non-acetaminophen induced acute liver failure and preliminary results suggest a modest but significant effect on survival in the early stages of ALF.16
The hepatoprotective effect of SAMe is, in part, associated hepatic GSH replenishment.11 SAMe donates its methyl group to a large variety of acceptor molecules including phospholipids and DNA. These reactions help to stabilize membranes (and thus maintain mitochondrial GSH) and control transcription events. In monocytes, SAMe decreases the production of pro-inflammatory cytokines (via inhibition of NK-κβ activation) and increases anti-inflammatory cytokine (IL-10 and IL-6) production. SAMe's anti-apoptotic may be related to the production of methylthioadenosine (MTA) through the aminopropylation pathway. While SAMe is anti-apoptotic in normal cells, it can be pro-apoptotic in hepatic cancer cell lines. This pro-apoptotic effect is likely related to the lack of different arms in the methionine metabolism pathway in cancer cells. For example, decreased reconversion of homocysteine to methionine can result in homocysteine induced endoplasmic reticulum stress mediated apoptosis. Accumulation of S-adenosylhomocysteine can also sensitize hepatocytes to TNF mediated apoptosis and LPS injury. These studies point to the important role of remethylation of homocysteine in patients receiving SAMe therapy. These pathways require betaine, B12 and folate as co-factors.
The cytoprotective effects of ursodeoxycholate (URSO) can be broadly grouped into 3 categories.17 First it promotes choleresis by: 1) a direct effect on biliary epithelium to promote the secretion of bicarbonate rich bile, and 2 ) mobilization of intracellular endosomal pools of transporters to the plasma membrane. This latter effect is mediated by activation of protein kinase C. URSO's anti-apoptotic activity is centered on maintenance of mitochondrial membrane integrity and involves activation of the PI3K/Akt and the ERK 1/2 pathways in hepatocytes. URSO prevents movement of BAX to mitochondria, mitochondrial ROS generation and promotes maintenance of mitochondrial GSH stores. URSO can decrease transcriptional activation of pro-apoptotic p53 signaling by increasing direct interaction of p53 with its cytoplasmic repressor, mdm-2.18 Evidence also suggests that UCDA can work as a biological response modifier of the glucocorticoid receptor (GR). UDCA can activate the GR by interacting with a distinct region of the receptor (not the same binding site as cortisol), induce nuclear translocation of the GR and suppress transcription of inflammatory mediators.19
Silymarin is a collection of flavinoids with proven hepatoprotective potential.20,21 Silymarin has anti-oxidant, anti-fibrotic, immunomodulatory/anti-inflammatory and growth promoting activity and modulates hepatocyte transport. Silymarin inhibits lipid peroxidation by acting as a free radical scavenger as well as helping to restore hepatic GSH levels. Silymarin interferes with the uptake of phalloidin (Amanita mushroom poisoning) and promotes the maintenance of biliary transporters in the plasma membrane. By enhancing RNA-polymerase activity and the transcription of rRNA silymarin promotes protein synthesis and hepatic regeneration. Silymarin is also an immunomodulator as it decreases lymphocyte cytotoxicity, increases IL-10 production, decreases pro-inflammatory cytokines production and inhibits lipooxygenase, NK-κβ and TNF signaling. Silymarin, a very lipophilic compound, can interact directly with biological membranes to prevent injury. In addition to it effects on hepatocytes, silymarin can prevent transforming growth factor-beta mediated activation of hepatic stellate cells into myofibroblasts and thus is anti-fibrotic.
Vitamin E is fat soluble and is primarily located within the phospholipid bilayer of the cell membranes where it has a major biological role in protecting polyunsaturated fats and other components of the cell membranes from oxidation by free radicals.22,23 The term "vitamin E" refers to a family of eight related, lipid-soluble, antioxidant compounds widely distributed in plants. Alpha-tocopherol is the most active form of vitamin E. In addition to it membrane protective effects, vitamin E analogues can modulate the activity of lipooxygenases and cyclooxygenases and protein kinase C and inhibit activation of NK-κβ.
1. Guicciardi ME, et al. Dig Liver Dis. 2002; 34:387.
2. Amaravadi RK, et al Clin Cancer Res. 2007 ; 13:7271.
3. Parcellier A, et al. Cell Signal. 2008; 20:21.
4. Li Z, Mizuno S, et al. Am J Physiol Gastrointest Liver Physiol. 2007;292:G639.
5. Spee B, Penning LC, et al. Comp Hepatol. 2005;7;4.
6. Spee B, et.al. Comp Hepatol. 2007; 31;6.
7. Cullen K, et.al. Am J Physiol Gastrointest Liver Physiol 2004; 287: G334.
8. Holz GG, et al. J Physiol. 2006;577:5.
9. Schwabe RF, et al. Gastroenterology. 2007; 132:2601.
10. Garcia-Ruiz C, et al. J Gastroenterol and Hepatol 2001;21:S3.
11. Mato JM, et al. Hepatology 2007;45: 1306.
12. Pinkse GGM, et al. Liver Int 2004; 24: 218.
13. Usechak P, et al. Hepatology 2007; 46: 282A .
14. Zafarullah M, et al. Cell Mol Life Scie 2003;60: 6.
15. Aitio ML Br J Clin Pharmacol. 2006; 61:5.
16. Lee WM, et al. Hepatology2007;46: 213A.
17. Beuers U. Nat Clin Pract Gastroenterol Hepatol 2006;3: 318.
18. Amaral JD, Castro RE, et al. J Biol Chem. 2007; 282:34250.
19. Solá S, et al. Hepatology. 2005;42:925.
20. Crocenzi FA, et al. Curr Med Chem. 2006;13:1055.
21. Pradhan SC, et al. Indian J Med Res. 2006;124:491.
22. Hickman I, et al. Hepatology. 2007;46:288.
23. Zingg JM. Clin Rev Med Chem 2007;7:543.