Activators of AMPK in the liver − Rakyat Biologi

AMPK is activated in response to a variety of metabolic stresses that typically, but not exclusively, change the cellular AMP:ATP ratio, either by increasing ATP consumption (activation of biosynthetic pathways) or reducing ATP production following hypoxia, glucose deprivation and inhibition of mitochondrial oxidative phosphorylation with metabolic poisons (arsenite, oligomycin, dinitrophenol, azide and antimycin A) (Towler and Hardie, 2007). AMPK plays a central role in the metabolic adaptation to acute and chronic nutritional stresses. For instance, AMPK is activated in the liver by the metabolic challenges imposed by either a 24-h fast (Munday et al., 1991, Witters et al., 1994) and dietary energy restriction (Jiang et al., 2008). However, in other studies, fasting and caloric restriction did not activate liver AMPK (Gonzalez et al., 2004, To et al., 2007). Interestingly, it has been noted that the metabolic demands during muscular work results in a decrease in energy status of the liver (Camacho et al., 2006). Indeed, increased AMPK activation has been demonstrated following short-term exercise in rat liver (Carlson and Winder, 1999, Park et al., 2002). Long-term exercise also induced significant increase in AMPK phosphorylation as well as AMPKa1- and a2-subunit mRNA levels in the liver, suggesting a role for hepatic AMPK in long-term exercise-induced hepatic adaptations (Takekoshi et al., 2006).

In the liver, the transition from the fasted to the fed state is also associated with physiological changes in energy dynamics. The reversal of the metabolic response to starvation includes alterations in enzyme phosphorylation states and changes in the concentration of key regulatory molecules. It has been reported that AMPK coordinates the changes in the activity and/or expression of a number of enzymes of lipid metabolism during refeeding (Munday et al., 1991, Gonzalez et al., 2004, Assifi et al., 2005, Dentin et al., 2005). Refeeding causes a 40% decrease in the activity of AMPKa1 within 1 h, with additional decrease in both AMPKa1 and AMPKa2 activities occurring between 1 and 24 h (Assifi et al., 2005). It is noteworthy that LKB1 activity could also be modulated during the starvation-refeeding transition in association with its acetylation and intracellular localization (Lan et al., 2008). The modification of AMPK activity during the starved-fed transition is compatible with earlier studies that linked these changes to increase in plasma insulin [reported to decrease AMPK activity in isolated hepatocytes (Witters and Kemp, 1992)], and decrease in glucagon [showed to activate hepatic AMPK (Sim and Hardie, 1988)], possibly by a protein kinase A-induced phosphorylation and activation of LKB1 (Kimball et al., 2004). In addition, changes in circulating levels of various hormones and adipokines during refeeding, may directly affect AMPK activity in the hepatocytes and could also contribute to the fasted-to-fed transition from catabolism to anabolism in the liver. It has been reported that hepatic AMPK can be regulated by ghrelin (Barazzoni et al., 2005, Kola et al., 2005), endocannabinoids (Kola et al., 2005), glucocorticoids (Christ-Crain et al., 2008), resistin (Banerjee et al., 2004) and adiponectin (Yamauchi et al., 2002) (Table 1).

As well as responding to metabolic stresses, hepatic AMPK activity is modulated by various pharmacological and natural drugs including [AICAR] (5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside) (Corton et al., 1995), compound A-769662 (Cool et al., 2006), polyphenols (Zang et al., 2006) and two major classes of existing antidiabetic drugs biguanides (metformin and phenformin) (Zhou et al., 2001) and thiazolidinediones (TZDs) (Saha et al., 2004).

AICAR is a cell-permeable nucleoside which could be metabolically converted to 5-aminoimidazole-4-carboxamide ribotide (AICA ribotide or ZMP) by adenosine kinase. ZMP shares some structural similarities with 5’-AMP and can mimic all of the allosteric effects of 5’-AMP on the AMPK system (Corton et al., 1995). During the last decade, AICAR has been extensively used both in vitro and in vivo to activate hepatic AMPK (Viollet et al., 2006) because it was generally assumed that activation of AMPK by AICAR does not affect cellular levels of AMP, ADP or ATP (Corton et al., 1995). However, this view has been recently challenged, showing that treatment of hepatocytes with AICAR concentrations above 200 mM depleted intracellular ATP levels (Guigas et al., 2006, Mukhtar et al., 2008). Importantly, AMPK-independent effects of AICAR in the control of hepatic glucose uptake (Guigas et al., 2006), phosphatidylcholine synthesis (Jacobs et al., 2007) and autophagic proteolysis (Meley et al., 2006) probably associated with its effect on ATP depletion where also recently reported. Detrimental effect of AICAR is likely to be due to an AMPK-independent inhibition of mitochondrial oxidative phosphorylation induced by a concomitant effect of ZMP on the mitochondrial respiratory chain complex 1 and a drop of adenine nucleotides and inorganic phosphate following its phosphorylation (Guigas et al., 2007). Furthermore, we established that ZTP accumulation induced uncoupling of mitochondrial oxidative phosphorylation, an effect that could worsen the change in cellular energetic by decreasing the yield of ATP synthesis (Guigas et al., 2007). In addition, it should be noted another important caveat in the use of AICAR because ZMP accumulation does affect certain other AMP-regulated enzymes such as glucokinase and fructose 1,6-bisphosphatase causing inhibition of glycolysis and gluconeogenesis in hepatocytes (Vincent et al., 1992, Vincent et al., 1991). Therefore, like all pharmacological approaches, results of experiments using AICAR must be interpreted with caution and it remains to be clearly determined whether all AICAR effects described are mediated by AMPK. In addition, it has been demonstrated that uptake of AICAR into cells, mediated by the adenosine transport system, is blocked by a number of protein kinase inhibitors, thus preventing ZMP accumulation and subsequent AMPK activation (Fryer et al., 2002, Guigas, unpublished results).

Recently, a new class of AMPK activators have been identified after the screening of a chemical library using partially purified AMPK from rat liver. The non nucleoside thienopyridone A-592017 emerged from the initial screen, and after optimization the more potent one, A-769662, was developed. Specificity of this new compound has been tested on a panel of 76 protein kinases and the majority of kinases were not significantly affected at 10 µM (Goransson et al., 2007) suggesting that A-769662 is a new specific activator of AMPK. Unlike other AMPK activators, A-769662 directly activates native AMPK complex purified from rat liver in cell-free essays by mimicking both effects of AMP on allosteric activation and inhibition of dephosphorylation of AMPK complex (Goransson et al., 2007, Sanders et al., 2007). However, A-769662 and AMP binding sites on AMPK complex are unlikely to be identical. Firstly, in the presence of saturating AMP concentration, A-769662 stimulated further AMPK activity (Cool et al., 2006, Goransson et al., 2007). Second, A-769662 allosterically activates AMPK complexes harboring a mutation in the g1 subunit that abolishes allosteric activation by AMP (Sanders et al., 2007). Interestingly, an AMPK complex lacking the glycogen binding domain of the b subunit or containing a mutation of Ser-108 to alanine (an autophosphorylation site within the glycogen binding domain of the b1 subunit) completely abolished the allosteric effect of A-769662, while only partially reducing AMP activation (Sanders et al., 2007). It has been reported that AMPK activation by A-769662 was independent of the upstream kinase utilized (Goransson et al., 2007, Sanders et al., 2007). Importantly, neither change in adenine nucleotide levels (Cool et al., 2006) nor alterations in mitochondrial oxidative phosphorylation (Guigas et al., 2008) following treatment with A-769662 have been detected in hepatocytes. Addition of A-769662 to primary mouse hepatocytes stimulates AMPK activity and phosphorylation of its known downstream targets but was completely abolished in hepatocytes lacking AMPKa1 and a2 catalytic subunits (Goransson et al., 2007). Short-term in vivo treatment with this compound recapitulates many of the effects expected for an hepatic activation of AMPK (Foretz et al., 2005) as it is mainly targeted to the liver (Cool et al., 2006).

Polyphenols, including resveratrol and epigallocatechin-3-gallate (EGCG), have been recently identified as potent activators for AMPK in vitro and in vivo (Baur et al., 2006, Collins et al., 2007, Zang et al., 2006). Some of the beneficial metabolic actions of polyphenols are mediated by their ability to activate sirtuin 1 (SIRT1), a mammalian ortholog of Sir2 (silent information regulator 2), an NAD-dependent deacetylase that acts as a master metabolic sensor of NAD+ and modulates cellular metabolism and life span (Baur et al., 2006). It has been demonstrated that SIRT1 activation by resveratrol functions as an upstream regulator in the LKB1/AMPK signaling axis (Hou et al., 2008). The ability of resveratrol to stimulate AMPK was mimicked by overexpression of SIRT1 and abolished by knockdown or pharmacological inhibition of SIRT1. Furthermore, AMPK activation by resveratrol-activated SIRT1 is mediated by the upstream kinase, LKB1, but not CaMKKb. The mechanism by which SIRT1 activation by polyphenols leads to LKB1-dependent AMPK activation relies on the direct deacetylation of LKB1 by SIRT1 (Lan et al., 2008). Indeed, activation of SIRT1 deacetylates LKB1, which in turn increases its cytoplasmic localization, its association with the LKB1 activator STRAD and its ability to activate AMPK. Nevertheless, it should be noted that AMPK activation by polyphenols may involve distinct regulators. It has been shown that the EGCG-induced increase in AMPK phosphorylation is mediated by CaMKK activation through production of ROS (Collins et al., 2007). Furthermore, the SIRT1 regulation of LKB1/AMPK signaling appears to be tissue-specific as resveratrol-stimulated AMPK activation in neurons was independent of SIRT1 (Dasgupta and Milbrandt, 2007).

The molecular pathway of AMPK activation by the antidiabetic drug metformin has been unclear (Zhou et al., 2001) but the direct inhibition of the respiratory chain complex 1 by the biguanide constitutes the more convincing molecular mechanism, thus, leading to reduced cellular ATP and increased AMP (El-Mir et al., 2000, Brunmair et al., 2004, Owen et al., 2000). This is supported by the fact that metformin treatment in primary hepatocytes resulted in a decrease in ATP content (Guigas et al., 2006). It should be noted that metformin could also exert AMPK-independent effects in the liver, probably due to its effect on cellular ATP levels (Guigas et al., 2006). It has been reported that TZDs acutely activate AMPK by a mechanism independent of PPARg-regulated gene transcription, which appears to be associated with change in cellular energy state and could potentially increase AMPK activity (Brunmair et al., 2004, Saha et al., 2004).