A Novel Mechanism of Starvation-Stimulated Hepatic Autophagy: Calcium-Induced O-GlcNAc-Dependent Signaling

Two central functions of the lysosomal degradative pathway of macroautophagy (hereafter referred to as autophagy) are to provide cells with metabolic substrates for energy generation in times of limited nutrients and act as a quality control mechanism to remove damaged or aged cellular components. Recent investigations have demonstrated that both functions regulate normal hepatic physiology and when impaired contribute to the pathophysiology of liver disease.⁽¹⁾ The full spectrum of autophagy’s metabolic functions in the liver has recently begun to be defined. The realization that insulin increases and glucagon decreases cellular levels of both autophagy and lipid breakdown led to the discovery that autophagy regulates hepatocyte lipid content and the development of hepatic steatosis.⁽²⁾ These findings were the first to link the metabolic functions of hepatic autophagy with liver disease development. These investigations and subsequent studies point to the importance of a better understanding of the function of hepatic autophagy in systemic metabolic control. Although numerous studies have examined the negative regulation of autophagy by insulin in the liver, little has been known about the mechanism(s) by which glucagon induces autophagy. The recent article Ruan H-B, Ma, Y, Torres, S, et al. Calcium-dependent O-GlcNAc signaling drives liver autophagy in adaptation to starvation. Genes Dev 2017;31:1655–1665 provides new insights into glucagon regulation of hepatic autophagy during nutrient deprivation. The findings demonstrate a novel function for the protein modification of O-linked β-N-acetylglucosamine (O-GlcNAc) in the regulation of starvation-induced autophagy through an effect on Unc-51-like kinase 1 (ULK1) which initiates the process of phagophore formation.⁽³⁾

In times of adequate nutrient supply glucose-induced insulin and amino acids trigger mTOR activation to suppress autophagy. With nutrient insufficiency, low serum glucose leads to pancreatic release of the counterregulatory hormone glucagon which increases hepatocyte autophagy. Induction of hepatic autophagy by this hormone is critical to maintaining whole body nutrient and energy balance through the generation of amino acids, glucose and free fatty acids.

The studies by Ruan, et. al.,⁽⁴⁾ specifically examined the role of the post-translational protein modification in which O-GlcNAc moieties are attached to serine or threonine residues. O-GlcNAcylation had been implicated previously as a nutrient sensor.⁽⁵⁾ The levels of O-GlcNAcylation generally positively correlate with the availability of glucose and free fatty acids, especially in chronic settings. However, up regulation of O-GlcNAcylation occurs in certain cell types with the deprivation of glucose in culture or food in mice. Employing mice with decreased hepatic autophagy from an adenoviral knockout of the autophagy gene Atg5, the authors confirmed that glucagon regulation of blood glucose levels during starvation was dependent on liver autophagy. Although the knockout employed was not hepatocyte specific raising the possibility of effects on other cell types, the in vivo findings were confirmed in cultured primary hepatocytes.

To prove mechanistic involvement of O-GlcNAcylation, the authors capitalized on the fact that the enzymes O-GlcNAc-transferase (OGT) and O-GlcNAcase (OGA) mediate the addition and removal of O-GlcNAc, respectively. Pharmacological inhibition of OGA in cultured hepatocytes and in vivo studies in Ogt knockout mice demonstrated that O-GlcNAcylation was critical for the induction of autophagy in response to starvation. Serum levels of amino acids, glucose and free fatty acids were all reduced in the knockout mice. Knockout mice also lost the ability to increase hepatic autophagy and glucose production in response to glucagon. Together these data demonstrate a new function for O-GlcNAcylation in controlling the autophagy-dependent supply of nutrients during starvation. The results also further establish the importance of hepatic autophagy in the regulation of metabolism in the whole animal.

Through a series of careful studies the authors identified the upstream activators and downstream effectors of glucagon’s effect on autophagy. Glucagon increased hepatocyte cytosolic Ca²⁺ levels through an effect on the Ca²⁺ channel InsP3R type 1 which controls calcium release from intracellular organelles such as the endoplasmic reticulum (ER). Calcium activated the kinase CaMKIIγ which phosphorylated OGT to increase O-GlcNAcylation and autophagy. The downstream target of O-GlcNAcylation was identified as ULK1 which when O-GlcNAcylated phosphorylated Beclin 1 to induce phagophore formation. ULK1 is positively regulated by AMP-activated protein kinase (AMPK) phosphorylation. O-GlcNAcylation increased AMPK association with and phosphorylation of ULK1 indicating that O-GlcNAcylation promoted the recruitment of AMPK to ULK1 to increase autophagy. One limitation of this study is that the mechanistic investigations of ULK1 were performed in nonhepatic transformed cell lines, and the findings should be confirmed in cultured hepatocytes and mouse liver. Nonetheless, the findings provide a very complete picture of the series of intracellular events that transduce the starvation-induced pancreatic release of glucagon into an increase in hepatic autophagy (Fig. 1). The work also establishes a new pathway for ULK1 activation that opposes the actions of insulin and amino acid induction of mTOR to inhibit autophagy.

FIG. 1.

Pathway by which starvation induces a glucagon-mediated increase in liver autophagy. Starvation lowers blood glucose levels leading to release of glucagon from the pancreas. Glucagon binds to receptors on the hepatocyte to trigger calcium release into the cytosol from intracellular organelles such as the endoplasmic reticulum by activation of the calcium channel InsP3R1. The increase in intracellular Ca²⁺ activates CaMKII by phosphorylation, and this kinase in turn phosphorylates and activates OGT. OGT activation triggers ULK1 O-GlcNAcylation which promotes ULK1 association with AMPK and ULK1 phosphorylation. Activated ULK1 phosphorylates Beclin 1 to initiate phagophore formation and increase autophagy. Increased autophagy supplies substrates including amino acids, glucose and free fatty acids (FAA) to maintain cellular energy levels in the setting of starvation. This nutritional up regulation of autophagy by glucagon may also serve to increase the other beneficial effects of autophagy in the hepatocyte such as resistance to cell injury and death.

The findings highlight both the importance of calcium signaling to hepatic autophagy and the intricacy of calcium regulation of hepatocellular autophagic function. Prior studies examining the inhibitory effects of saturated acids on hepatic autophagy demonstrated that palmitate-induced ER stress decreases activity of the sarco-ER calcium ATPase (SERCA) which regulates cellular calcium homeostasis by sequestering calcium in the ER.⁽⁶⁾ Palmitate-inhibited SERCA function led to increased intracellular calcium that blocked autophagosome-lysosome fusion to decrease autophagic function, the opposite finding for increased cytosolic calcium from the present study. These opposing findings need to be reconciled but may represent different physiological effects of a transient (glucagon) versus sustained (ER stress) increase in intracellular calcium or of the spatial localization of the increase in cytosolic calcium.

An unanswered question is whether O-GlcNAcylation regulates hepatic autophagy strictly during times of nutrient depletion or also in response to other hepatic stresses. Culturing cells in a simple salt solution in the absence of glucagon was sufficient to activate the CaMKII-OGT-ULK1 pathway indicating that activation is not dependent solely on glucagon. Induction of hepatic autophagy is an important protective pathway in a number of forms of liver injury including that from hepatotoxins.⁽¹⁾ Autophagy limits mouse hepatic injury from galactosamine/lipopolysaccharide and acetaminophen through a variety of mechanisms including the down regulation of caspase activation and removal of protein adducts, respectively.⁽¹⁾ A recent report has indicated that hyper-O-GlcNAcylation promotes acetaminophen-induced liver injury which is contrary to the expected beneficial effect of this post-translational modification on autophagy, but may be explained by autophagy-independent effects on glutathione biosynthesis.⁽⁷⁾ This finding together with the current study suggests that maintaining O-GlcNAcylation at an optimal level is required for hepatic homeostasis.⁽⁸⁾ The varied functions of O-GlcNAcylation which may vary depending on its cellular levels make it uncertain as to whether this pathway would be a viable therapeutic target to increase autophagy in disease states.

The recognition that hepatic autophagic function is impaired in liver disease, and that defects in autophagy may underlie disease pathophysiology, has suggested that agents that increase autophagy may be an effective therapy in liver disease.⁽¹⁾ An initial trial of the autophagy-inducing drug carbamazepine in the treatment of liver disease from α₁-antitrypsin deficiency is in progress (ClinicalTrials.gov, NCT01379469). By providing insights into the mechanisms by which autophagy can be induced in the liver, this study and others are important to the potential discovery of new therapeutic targets that may be exploited in this pathway for the treatment of liver disease.