G6Pase-beta is an enzyme catalyzing the hydrolysis of G6P to glucose. To date, 33 separate G6Pase-beta mutations have been identified in GSD-Irs patients but only the p.R253H and p.G260R missense mutations have been characterized functionally for pathogenicity. We now functionally characterized 16 of the 19 known missense mutations using a sensitive assay, based on a recombinant adenoviral vector-mediated expression system, to demonstrate pathogenicity. A database of residual enzymatic activity retained by the G6Pase-beta mutations will serve as a reference for evaluating genotype-phenotype relationships. We have shown that G6pc-/- mice receiving rAAV8-G6PC-mediated gene transfer that express 5 units of hepatic G6Pase-alpha activity (AAV mice) maintains glucose homeostasis, shows no evidence of hepatocellular adenoma (HCA)/carcinoma (HCC), and are pretected against age-related insulin resistance or obesity. We undertook studies to delineate the molecular mechanisms underlying this beneficial metabolic phenotype. Studies have shown that mice over-expressing hepatic ChREBP exhibit improved glucose and lipid metabolism, resulting from Akt activation and increased expression of stearoyl-CoA desaturase 1 (SCD1). In AAV mice, elevated concentrations of hepatic G6P stimulate hepatic ChREBP signaling, leading to increased SCD1, and active p-Akt-S473 and p-Akt-T308, consistent with activation of ChREBP signaling as one mechanism underlying the beneficial phenotype. We further show that the AAV mice mimic animals living under calorie restriction (CR). The major downstream modulators of CR are AMPK and sirtuin-1 (SIRT1), a NAD+-dependent deacetylase. We showed that hepatic NAD+ concentrations in the AAV mice are higher than the age-matched control mice, consistent with SIRT1 activation. The AAV mice also exhibit increased protein levels of AMPK and active p-AMPK-T172. Aging is also associated with mitochondrial dysfunction, so protection of mitochondria may also contribute to the observed phenotype. The PGC-1 is a master regulator of energy metabolism and mitochondrial biogenesis, playing key roles in the maintenance of mitochondrial integrity, biogenesis, and function. We showed that the expression of PGC-1 is up-regulated in the AAV mice. Moreover, protein levels of complex I to V of the mitochondrial electron transport chain are up-regulated in AAV mice. Taken together, the underlying mechanisms responsible for the beneficial metabolic phenotype correlate with activation of ChREBP and AMPK/SIRT1/PGC-1 signaling pathways in the livers of AAV mice. The hallmarks of GSD-Ia are impaired glucose homeostasis and long-term risk of HCA)/HCC. The predominant subtypes of HCA in GSD-Ia are inflammatory HCA and -catenin-mutated HCA. We have shown that AAV mice that express 5 units of hepatic G6Pase- activity maintain glucose homeostasis, lack HCA/HCC, and live under CR. We now show that 75% of rAAV-treated mice expressing 1.5-4.9 units of hepatic G6Pase- activity (AAV-low-NT mice) exhibit a similar phenotype. We examined pathways involved in CR for insights into mechanisms underlying the absence of HCA/HCC in AAV/AAV-low-NT (AAV-NT) mice. The CR mediators AMPK and SIRT1, which share activators, actions, and targets, were activated in AAV-NT mice. AMPK elicits anti-inflammatory action by inhibiting phosphorylation and activation of STAT3, a transcription factor that promotes cancer-promoting inflammation. SIRT1 represses NFB activity via deacetylates of NFB-p65, a transcription factor that promotes inflammation-associated cancer. The signaling by STAT3 and NF-B is highly interconnected. Together they regulate multiple genes involved in tumor proliferation, survival, and invasion. In AAV-NT mice, hepatic levels of active p-STAT3-Y705 and ac-NFB-p65-K310 were reduced. SIRT1 also inhibits cancer metastasis. We showed that the expression of mesenchymal markers, STAT3 targets, NFB targets, and -catenin targets, all of which promote tumorigenesis, were also reduced. AAV-NT mice also expressed increased levels of E-cadherin, FGF21, and the tumor suppressor -klotho. Importantly, treating AAV mice with a SIRT1 inhibitor EX-527 markedly reversed many of the anti-tumorigenic pathways. In summary, activation of hepatic AMPK/SIRT1 and FGF21/-klotho signaling combined with down-regulation of STAT3 and NFB signaling underlie the absence of HCA/HCC in AAV-NT mice. The most severe complications in GSD-Ia are the development of HCA/HCC of unknown etiology. The global G6pc-/- mice die early, well before HCA/HCC can develop, making mechanism studies of HCA/HCC difficult. We therefore generated the liver-specific G6pc knock-out mice (L-G6pc-/-) that survive to adulthood and develop HCA. Using L-G6pc-/- mice, we investigated the underlying mechanisms of HCA/HCC in GSD-Ia. Studies have shown that autophagy-deficient mice develop HCA. We hypothesized that defective autophagy may underlie HCA/HCC development in GSD-Ia. Autophagy can be regulated directly by SIRT1 via deacetylation of autophagy-related proteins and indirectly via deacetylation and activation of FoxO factors which transactivate autophagy genes. SIRT1 activity can be activated by the cofactor NAD+ but levels of NAD+ were unchanged in L-G6pc-/- livers. The expression of SIRT1 can be suppressed by lipogenic factors such as ChREBP and PPAR-, and stimulated by FoxO1 and PPAR- in response to nutrient starvation. In G6pc-/- liver, activation of ChREBP signaling was associated with increased lipogenesis and suppressed PPAR- expression, leading to a marked increase in hepatic steatosis and elevated PPAR- expression. The net outcome was a decreased expression of SIRT1 in G6pc-/- livers, compared to controls. In addition, protein levels of FoxO3a were decreased in G6pc-/- livers. Collectively, reduced SIRT1-FoxO signaling appears to contribute to impaired autophagy in G6pc-/- livers. The G6Pase--deficient liver also displays mitochondrial dysfunction characterized by a reduction in oxidative phosphorylation, a decrease in overall mitochondrial numbers, and a decrease in functional mitochondria. The mechanism underlying mitochondrial dysfunction arises from down-regulation of hepatic SIRT1-PGC-1 signaling pathway. We further showed that the underlying mechanisms responsible for HCA/HCC formation in GSD-Ia include accumulation of p62 aggregates that promotes tumorigenesis and marked mitochondrial and oxidative DNA damage. Importantly, we showed that restoration of hepatic G6Pase- expression by rAAV- G6PC-mediated gene transfer normalizes autophagy deficiency, and restores SIRT1-FoxO signaling in L-G6pc-/- mice. Taken together, our study provides the underlying mechanisms for HCA/HCC in GSD-Ia, and also suggests that correction of defective autophagy by gene therapy or pharmacological intervention will prevent or slow down the chronic development of HCA/HCC in human GSD-Ia patients.
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