Molecular mechanism underlying impaired hepatic autophagy in glycogen storage disease type Ib.

Type Ib glycogen storage disease (GSD-Ib) is caused by a deficiency in the glucose-6-phosphate (G6P) transporter (G6PT) that translocates G6P from the cytoplasm into the endoplasmic reticulum lumen, where the intraluminal G6P is hydrolyzed to glucose by glucose-6-phosphatase-α (G6Pase-α). Clinically, GSD-Ib patients manifest a metabolic phenotype of impaired blood glucose homeostasis and a long-term risk of hepatocellular adenoma/carcinoma (HCA/HCC). Studies have shown that autophagy deficiency contributes to hepatocarcinogenesis. In this study, we show that G6PT deficiency leads to impaired hepatic autophagy evident from attenuated expression of many components of the autophagy network, decreased autophagosome formation and reduced autophagy flux. The G6PT-deficient liver displayed impaired sirtuin 1 (SIRT1) and AMP-activated protein kinase (AMPK) signaling, along with reduced expression of SIRT1, forkhead boxO3a (FoxO3a), liver kinase B-1 (LKB1) and the active p-AMPK. Importantly, we show that overexpression of either SIRT1 or LKB1 in G6PT-deficient liver restored autophagy and SIRT1/FoxO3a and LKB1/AMPK signaling. The hepatosteatosis in G6PT-deficient liver decreased SIRT1 expression. LKB1 overexpression reduced hepatic triglyceride levels, providing a potential link between LKB1/AMPK signaling upregulation and the increase in SIRT1 expression. In conclusion, downregulation of SIRT1/FoxO3a and LKB1/AMPK signaling underlies impaired hepatic autophagy which may contribute to HCA/HCC development in GSD-Ib. Understanding this mechanism may guide future therapies.

Correction of metabolic abnormalities in a mouse model of glycogen storage disease type Ia by CRISPR/Cas9-based gene editing.

Glycogen storage disease type Ia (GSD-Ia), deficient in glucose-6-phosphatase-α (G6PC), is characterized by impaired glucose homeostasis and a hallmark of fasting hypoglycemia. We have developed a recombinant adeno-associated virus (rAAV) vector-mediated gene therapy for GSD-Ia that is currently in a phase I/II clinical trial. While therapeutic expression of the episomal rAAV-G6PC clinical vector is stable in mice, the long-term durability of expression in humans is currently being established. Here we evaluated CRISPR/Cas9-based in vivo genome editing technology to correct a prevalent pathogenic human variant, G6PC-p.R83C. We have generated a homozygous G6pc-R83C mouse strain and shown that the G6pc-R83C mice manifest impaired glucose homeostasis and frequent hypoglycemic seizures, mimicking the pathophysiology of GSD-Ia patients. We then used a CRISPR/Cas9-based gene editing system to treat newborn G6pc-R83C mice and showed that the treated mice grew normally to age 16 weeks without hypoglycemia seizures. The treated G6pc-R83C mice, expressing ≥ 3% of normal hepatic G6Pase-α activity, maintained glucose homeostasis, displayed normalized blood metabolites, and could sustain 24 h of fasting. Taken together, we have developed a second-generation therapy in which in vivo correction of a pathogenic G6PC-p.R83C variant in its native genetic locus could lead to potentially permanent, durable, long-term correction of the GSD-Ia phenotype.

Mechanisms of endothelin-1-induced MAP kinase activation in adrenal glomerulosa cells.

G protein-coupled receptors (GPCRs) such as angiotensin II, bradykinin and endothelin-1 (ET-1) are critically involved in the regulation of adrenal function, including aldosterone production from zona glomerulosa cells. Whereas, substantial data are available on the signaling mechanisms of ET-1 in cardiovascular tissues, such information in adrenal glomerulosa cells is lacking. Bovine adrenal glomerulosa (BAG) cells express receptors for endothelin-1 (ET-1) and their stimulation caused phosphorylation of Src (at Tyr416), proline-rich tyrosine kinase (Pyk2 at Tyr402), extracellularly regulated signal kinases (ERK1/2), and their dependent proteins, p90 ribosomal S6 kinase (RSK-1) and CREB. ET-1 elicited these responses predominantly through activation of a G(i)-linked cascade with a minor contribution from the G(q)/PKC pathway. Whereas, selective inhibition of EGF-R kinase with AG1478 caused complete inhibition of EGF-induced ERK/RSK-1/CREB activation, it caused only partial reduction (30-40%) of such ET-1-induced responses. Consistent with this, inhibition of matrix metalloproteinases (MMPs) with GM6001 reduced ERK1/2 activation by ET-1, consistent with partial involvement of the MMP-dependent EGF-R activation in this cascade. Activation of ERK/RSK-1/CREB by both ET-1 and EGF was abolished by inhibition of Src, indicating its central role in ET-1 signaling in BAG cells. Moreover, the signaling characteristics of ET-1 in cultured BAG cells closely resembled those observed in clonal adrenocortical H295R cells. The ET-1-induced proliferation of BAG and H295 R cells was much smaller than that induced by Ang II or FGF. These data demonstrate that ET-1 causes ERK/RSK-1/CREB phosphorylation predominantly through activation of G(i) and Src, with a minor contribution from MMP-dependent EGF-R transactivation.

Differential pathways of angiotensin II-induced extracellularly regulated kinase 1/2 phosphorylation in specific cell types: role of heparin-binding epidermal growth factor.

Stimulation of the angiotensin II (Ang II) type 1 receptor (AT1-R) causes phosphorylation of extracellularly regulated kinases 1 and 2 (ERK1/2) via epidermal growth factor receptor (EGF-R) transactivation-dependent or -independent pathways in Ang II target cells. Here we examined the mechanisms involved in agonist-induced EGF-R transactivation and subsequent ERK1/2 phosphorylation in clone 9 (C9) hepatocytes, which express endogenous AT1-R, and COS-7 and human embryonic kidney (HEK) 293 cells transfected with the AT1-R. Ang II-induced ERK1/2 activation was attenuated by inhibition of Src kinase and of matrix metalloproteinases (MMPs) in C9 and COS-7 cells, but not in HEK 293 cells. Agonist-mediated MMP activation in C9 cells led to shedding of heparin-binding EGF (HB-EGF) and stimulation of ERK1/2 phosphorylation. Blockade of HB-EGF action by neutralizing antibody or its selective inhibitor, CRM197, attenuated ERK1/2 activation by Ang II. Consistent with its agonist action, HB-EGF stimulation of these cells caused marked phosphorylation of the EGF-R and its adapter molecule, Shc, as well as ERK1/2 and its dependent protein, p90 ribosomal S6 kinase, in a manner similar to that elicited by Ang II or EGF. Although the Tyr319 residue of the AT1-R has been proposed to be an essential regulator of EGF-R transactivation, stimulation of wild-type and mutant (Y319F) AT1-R expressed in COS-7 cells caused EGF-R transactivation and subsequent ERK1/2 phosphorylation through release of HB-EGF in a Src-dependent manner. In contrast, the noninvolvement of MMPs in HEK 293 cells, which may reflect the absence of Src activation by Ang II, was associated with lack of transactivation of the EGF-R. These data demonstrate that the individual actions of Ang II on EGF-R transactivation in specific cell types are related to differential involvement of MMP-dependent HB-EGF release.