A deficiency in glucose-6-phosphatase-α (G6Pase-α) in glycogen storage disease type Ia (GSD-Ia) leads to impaired glucose homeostasis and metabolic manifestations including hepatomegaly caused by increased glycogen and neutral fat accumulation. A recent report showed that G6Pase-α deficiency causes impairment in autophagy, a recycling process important for cellular metabolism. However, the molecular mechanism underlying defective autophagy is unclear. Here we show that in mice, liver-specific knockout of G6Pase-α (L-G6pc-/-) leads to downregulation of sirtuin 1 (SIRT1) signaling that activates autophagy via deacetylation of autophagy-related (ATG) proteins and forkhead box O (FoxO) family of transcriptional factors which transactivate autophagy genes. Consistently, defective autophagy in G6Pase-α-deficient liver is characterized by attenuated expressions of autophagy components, increased acetylation of ATG5 and ATG7, decreased conjugation of ATG5 and ATG12, and reduced autophagic flux. We further show that hepatic G6Pase-α deficiency results in activation of carbohydrate response element-binding protein, a lipogenic transcription factor, increased expression of peroxisome proliferator-activated receptor-γ (PPAR-γ), a lipid regulator, and suppressed expression of PPAR-α, a master regulator of fatty acid β-oxidation, all contributing to hepatic steatosis and downregulation of SIRT1 expression. An adenovirus vector-mediated increase in hepatic SIRT1 expression corrects autophagy defects but does not rectify metabolic abnormalities associated with G6Pase-α deficiency. Importantly, a recombinant adeno-associated virus (rAAV) vector-mediated restoration of hepatic G6Pase-α expression corrects metabolic abnormalities, restores SIRT1-FoxO signaling, and normalizes defective autophagy. Taken together, these data show that hepatic G6Pase-α deficiency-mediated down-regulation of SIRT1 signaling underlies defective hepatic autophagy in GSD-Ia.
The glucose-6-phosphatase (Glc-6-Pase) family comprises two active endoplasmic reticulum (ER)-associated isozymes: the liver/kidney/intestine Glc-6-Pase-␣ and the ubiquitous Glc-6-Pase-. Both share similar kinetic properties. Sequence alignments predict the two proteins are structurally similar. During glucose 6-phosphate (Glc-6-P) hydrolysis, Glc-6-Pase-␣, a nine-transmembrane domain protein, forms a covalently bound phosphoryl enzyme intermediate through His 176 , which lies on the lumenal side of the ER membrane. We showed that Glc-6-Pase- is also a nine-transmembrane domain protein that forms a covalently bound phosphoryl enzyme intermediate during Glc-6-P hydrolysis. However, the intermediate was not detectable in Glc-6-Pase- active site mutants R79A, H114A, and H167A. Using [ 32 P]Glc-6-P coupled with cyanogen bromide mapping, we demonstrated that the phosphate acceptor in Glc-6-Pase- is His 167 and that it lies inside the ER lumen with the active site residues, Arg 79 and His 114 . Therefore Glc-6-Pase-␣ and Glc-6-Pase- share a similar active site structure, topology, and mechanism of action.The glucose-6-phosphatase (Glc-6-Pase) 1 family is composed of three proteins: Glc-6-Pase-␣ (1-4), Glc-6-Pase- (5-7) (previously known as UGRP (ubiquitously expressed Glc-6-Pase related protein)), and islet-specific Glc-6-Pase-related protein (8,9). Whereas Glc-6-Pase-␣ and Glc-6-Pase- are functional phosphohydrolases, the islet-specific Glc-6-Pase-related protein lacks enzymatic activity.The prototype of the family, Glc-6-Pase-␣, is a 357-amino acid, nine-transmembrane domain, endoplasmic reticulum (ER)-associated protein (10, 11), which is expressed primarily in the liver, kidney, and intestine (12, 13). Glc-6-Pase-␣ catalyzes the hydrolysis of glucose 6-phosphate (Glc-6-P) to glucose in the terminal step of gluconeogenesis and glycogenolysis (13). Between meals, the resulting release of glucose to the blood maintains glucose homeostasis. Naturally occurring loss of function mutations in Glc-6-Pase-␣ cause glycogen storage disease type Ia, a disorder that is characterized by loss of blood glucose homeostasis and disorders of glycogen and lipid metabolism (reviewed in Refs. 14 and 15).Glc-6-Pase- is a ubiquitously expressed, 346-amino acid membrane protein that shares a 36% sequence identity to Glc-6-Pase-␣ (5-7). Despite the absence of any apparent ER retention motif, Glc-6-Pase- is also localized in the ER membrane (6), although its orientation in the membrane is not known. The subcellular localization of the Glc-6-Pase- active site is not known, although it is assumed to be similar to Glc-6-Pase-␣. Both Glc-6-Pase- and Glc-6-Pase-␣ couple with the Glc-6-P transporter to form an active Glc-6-Pase complex, and both share similar kinetic properties with respect to Glc-6-P hydrolysis (6).The active site of Glc-6-Pase-␣ was originally identified by the presence of a conserved phosphatase signature motif found in lipid phosphatases, acid phosphatases, and vanadium haloperoxidases (16,17). This motif was show...
Glucose-6-phosphatase (G6Pase), a key enzyme in glucose homeostasis, is anchored to the endoplasmic reticulum by nine transmembrane helices.
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