Induction of Autophagy by Starvation and Regulation by Amino Acids and Hormones.
The best-known autophagy inducer is food restriction or starvation. Under this condition, autophagy is activated to provide cells with the necessary nutrients through degradation of intracellular materials. In animals, starvation induces the largest protein loss in the liver. In the first 48 hours, mice and rats can lose 25%-40% of their liver protein.23 The loss seems to be mainly in the cytosol with most subcellular constituents except nuclei and DNA, resulting in a 25% reduction in cell volume in the first 24 hours of starvation.12 The involvement of macroautophagy and the lysosome was realized when protein loss was found to be associated with stimuli that cause autophagy, such as glucagon administration or deprivation of amino acids, insulin, or serum in the perfused rat liver and heart and in cultured cells.12 Amino acid deprivation has since been employed as a standard simulation for autophagic degradation of proteins in mammalian cells. In yeast, nitrogen deprivation is an equivalent stimulus for autophagy.24
Mortimore and Poso were largely responsible for the careful determination of the kinetics of protein degradation in the liver and the concept that long-term protein degradation is mediated by autophagy.12 For example, they found that the process of autophagy stimulated by amino acid deprivation in the liver was rapid, with the autophagosome having a half-life of only 6-9 minutes.12 The turnover rate of the autophagosome is well correlated with the proteolysis rate at different amino acid levels. Many factors could suppress autophagosome formation, including amino acids, insulin, cycloheximide, and 3-methyladenine (3-MA). Together these data have established that macroautophagy can account for the accelerated proteolysis in the liver on amino acid deprivation.
About 8 so-called regulatory amino acids (Leu, Tyr, Phe, Gln, Pro, Met, Trp, and His) could suppress macroautophagy in perfused rat liver at 0.5× and 4× the normal plasma concentration, among which leucine is by far the most effective.12 Leucine is also the only amino acid that can inhibit protein degradation in myocytes and adipocytes. Alanine has a coregulatory effect, in that it could work with regulatory amino acids at 1× the normal plasma concentration to achieve the maximal inhibitory effect but has no impact on proteolysis by itself. The coregulatory effect of alanine could also be mimicked by insulin.
Glucagon was perhaps the first hormone known to simulate autophagy. Ashford and Porter reported “autophagy-like” structures in glucagon-perfused rat liver in 1962.8 Soon it was confirmed that glucagon can stimulate autophagy in the liver.9, 25 The glucagon receptor is G-protein-coupled. The effector of the coupled Gαs is adenylyl cyclase. Thus, cAMP and the downstream protein kinase A mediate the effects of glucagon. Consistently, β-agonists such as epinephrine, which also activates adenylyl cyclase, and cAMP itself have effects similar to glucagon on autophagy induction. Interestingly, their autophagic effect is liver specific. In the muscle they instead inhibit autophagy and protein degradation.12
Glucagon-induced autophagic proteolysis in the liver does not seem to be related to glycogenolysis, which seems to be different from its role in glycogen autophagy (see below). Consistently, neither glucose nor fatty acids have a regulatory role in autophagic protein degradation in the liver.12 It is not clear how glucagon stimulates autophagy in the liver, but it could be related to the regulatory pathway of amino acids. Thus, it could reverse the inhibitory effect of regulatory amino acids at 0.5× the normal plasma concentration to induce autophagy, and its ability to induce autophagy reaches the maximal level at the normal amino acid concentration. However, its autophagic effect could be blocked by amino acids at twice the normal plasma level.26
Very early on, insulin was found to have an inhibitory effect on autophagy in the liver.27, 28 Testing the effect of insulin on autophagy was initially based on the desire to search for antagonists of glucagon and on the observation that insulin could correct the abnormally elevated autophagy level in livers of severely diabetic rats. Interestingly, the inhibitory activity of insulin is universal and can be also observed in cardiac and skeletal muscles, kidney, adipose tissue, and cultured cells despite that the maximal effect varies and the degradation kinetics differ in these tissues.12 The inhibitory effect is likely related to the property of insulin as a growth factor and is mediated by the class I PI-3 kinase and PKB/Akt, which results in activation of mTOR (Fig. 3).29 Suppression of autophagy by class I PI-3 kinases had been demonstrated earlier in a colon cancer cell line.30, 31 In isolated rat hepatocytes, insulin can stimulate mTOP-dependent p70 S6 kinase, leading to increased S6 phosphorylation.29 Rapamycin can block insulin-induced S6 phosphorylation and reverse insulin-induced autophagy suppression, suggesting that insulin's activity can be mediated by mTOR.29 The importance of TOR in suppressing autophagy has been shown in yeast, in which suppression of TOR with rapamycin can induce autophagy even in nutrient-replete conditions.32
The mechanisms by which the regulatory amino acids suppress autophagy seem to be more complicated and have not been worked out. In isolated rat hepatocytes, amino acids are potent activators of p70 S6 kinase via mTOR.29, 33 However, unlike insulin, rapamycin was shown to have no29 or only a partial33 effect on the antiautophagy activity of amino acids despite being able to block amino acid–induced S6 phosphorylation. It thus seems that amino acids may have distinct functions in meeting nutrient demands, which are mediated by mTOR to promote protein synthesis, and in suppressing the catabolic autophagy pathway, which is likely to be independent of mTOR, at least in hepatocytes.
How could amino acids fulfill these 2 functions at the same time? One clue may come from the finding that activation of mTOR by amino acids is not through the traditional class I PI-3 kinase pathway and is thus independent of PKB/Atk, TSC1/TSC2, and RheB (Fig. 3).34 Rather, it is mediated by the class III PI-3 kinase, VPS34.34 But VPS34, together with its regulatory component, VPS15, is also known to be involved in autophagy induction in both yeast and mammalian cells. The substance 3-methyladenine was the first known suppressor of autophagic proteolysis as demonstrated in isolated hepatocytes35 and was subsequently shown to be a PI-3 kinase inhibitor.36 The importance of the class III PI-3 kinase (VPS34), but not the class I PI-3 kinase, which actually has opposite effects, in promoting autophagy was then demonstrated in a colon cancer cell line30 and in yeast.37 In yeast, VPS34 forms 2 different complexes, with complex I (containing Atg6/Beclin 1 and Atg14) functioning in the autophagy pathway. Thus, it is possible that amino acids may direct VPS34 from the catabolic autophagy pathway to the synthetic mTOR pathway, resulting in suppression of the former and activation of the latter. Neither the mechanism by which amino acids activate VPS34 nor how VPS34 subsequently turns on mTOR is known at the present time. But revealing these mechanisms would likely lead to the resolution of how VPS34 may possess opposite functions.
Amino acids could employ other mechanisms to suppress autophagy. Recently it has been found that both insulin and amino acids, such as phenylalanine, could also work through a pathway related to Gαi3 because deletion of Gαi3 eliminates their protective effects against protein degradation in perfused liver.38 Gαi3 is a Gα subunit of heterotrimeric G proteins, and its positive role (in guanosine diphosphate [GDP]-binding status) in autophagy induction was shown previously in a colon cancer cell line.39 Consistently, an early study implicated the involvement of guanosine triphosphatase (GTPase) activity in autophagy induction in permeabilized hepatocytes.40 How insulin and amino acids affect Gαi3 and how Gαi3 in turn affects autophagy are not known. In other types of cells, it has been found that amino acids may modulate activation of Raf-1 kinase and therefore reduce extracellular signal-regulated kinase 1/2 (ERK1/2) activation, which in turn reduces the phosphorylation and activity of GAIP.41, 42 GAIP is a GTPase-activating protein (GAP) for Gαi3 and elevates its GTPase activity. As such, GAIP promotes the GDP status of Gαi3 and is proautophagic. There is also some evidence suggesting that the Gαi3-mediated mechanism may interface with the VPS34-mediated mechanism, because activation of the latter could rescue autophagy caused by the defect in the former.30 It will be interesting to determine how these events are intertwined.
Effect of Energy Level and AMPK in Autophagy Induction in Hepatocytes.
Early studies have shown that the autophagy process is ATP dependent during both the sequestration and postsequestration steps.5 Thus, depletion of ATP will hamper autophagy. However, direct suppression of energy supply may have complicated outcomes. An obvious target of such manipulations is adenosine monophosphate-activated protein kinase (AMPK), which is activated by a high AMP/ATP ratio and in turn switches off ATP-dependent processes. An initial study found that an analog of AMP, AICAR, suppressed autophagic sequestration of lactose dehydrogenase in isolated hepatocytes.43 Although this suppressive effect of AMPK indicated by the use of AICAR as well as metformin, another indirect AMPK activator, could be confirmed in both hepatocytes and nonhepatocytes, complementary approaches employing an AMPK inhibitor, compound C, or a dominant-negative AMPK mutant instead indicate that AMPK is required for autophagy induced by amino acid deprivation.44 This latter finding that AMPK is a positive regulator of autophagy is supported by other studies in yeast45 and in mammalian cells46, 47 under various conditions, including ischemia. In general, these latest observations are consistent with the dogma that AMPK can suppress TOR and TOR suppresses autophagy (Fig. 3).
Thus, the suppressive effects of AICAR and metformin on nutrient deprivation-induced autophagy in hepatocytes may not be related to their effects on AMPK.44 Indeed, the AMPK-independent effects of AICAR have been observed in other cases.48, 49 Furthermore, this suppressive effect may be cell-type specific because in both murine embryonic fibroblasts and HCT116 colon cancer cells, AICAR and metformin are able to induce autophagy in a p53-dependent manner.50
Glycogen Autophagy and Neonatal Survival.
Autophagy activity is essential for the survival of newborn animals. Within 1 day of birth, genetic deletion of Atg5 or Atg7 causes the death of neonatal mice, which can be rescued by forced milk feeding.51, 52 In the mouse, based on changes in the subcellular localization of Atg8/LC3, it could be determined that autophagy induction increases in the first half hour after birth, peaks at 3-6 hours, and returns to baseline level by 2.5 days after birth in the liver, heart, diaphragm, gastrocnemius muscle, lung, and pancreas.51 Autophagy seems to be required for providing the necessary nutrients from the stored cellular supply during this critical period as the newborn adapts to the nutrients from the milk.
Although degradation of cellular proteins by autophagy may be required to maintain plasma amino acids level in the newborn, which is lower in Atg5- or Atg7-deficient mice,51, 52 amino acids may not be the major nutrient required for survival. What is required most immediately after birth is probably glucose for such energy-demanding organs as the heart and diaphragm. Indeed, Atg5-deficient mice have increased AMPK activation in the heart, suggesting a shortage in ATP production.51 However, gluconeogenic mechanisms are not fully established in the newborn,53 and they may need a direct supply of glucose. Degradation of glycogen could meet this demand.
Glycogen can be found in 2 spatially separated cellular locations in hepatocytes, the cytosol (hyaloplasmic pool), and autophagosomes. Autophagy actively participates in the overall breakdown of cellular glycogen and can selectively degrade polysaccharides. Autophagic degradation of glycogen in the newborn liver has long been recognized as an important survival mechanism for the newborn to adapt to the postnatal environment.53 Glycogen autophagy is likely stimulated by glucagon, which is secreted in response to hypoglycemia normally occurring in the newborn. In newborn hepatocytes, most of the autophagosomes are spatially and functionally related to glycogen, appearing at the borders of glycogen stores and usually containing various amounts of glycogen.54 Acid glucosidase in the lysosome is a major glycogen-hydrolyzing enzyme, and its activity increases rapidly after birth in both the liver and the heart, peaking at 6 hours in the livers of newborn rats, which is coincident with the abundance of autophagosomes around the glycogen stores. These early kinetic observations seem to be remarkably consistent with the new findings using GFP-LC3.51 This increased glycogen-hydrolyzing activity seems to be relatively specific, as there are no changes in other lysosomal enzyme activities in the liver or heart at this time of life. On the other hand, glycogen autophagy is also seen in the heart and muscles of the newborn.
Glycogen autophagy in the liver seems to be regulated by the same mechanisms already explained for protein degradation. It can thus be stimulated by glucagon, cAMP, β-adrenergic agonists, or rapamycin but suppressed by β-antagonists or insulin.53 Uniquely, glycogen autophagy could be suppressed by parenteral glucose. It is not clear how the relative selectivity of glycogen degradation versus protein degradation under the same hormonal regulation is achieved. In addition, because glucagon can promote breakdown of hyaloplasmic glycogen via the action of glycogen phosphorylase, how autophagic degradation of glycogen is coordinated with nonautophagic degradation is not clear. Perhaps the unique neonatal cellular milieu provides conditions favoring glycogen autophagy, which may be a more efficient or a more rapid way to produce nonphosphorylated glucose to the hungry newborn.