Hepatic mitochondrial dysfunction is a feature of Glycogen Storage Disease Type Ia (GSDIa)

Abstract

Glycogen storage disease type Ia (GSDIa, von Gierke disease) is the most common glycogen storage disorder. It is caused by the deficiency of glucose-6-phosphatase, an enzyme which catalyses the final step of gluconeogenesis and glycogenolysis. Clinically, GSDIa is characterized by fasting hypoglycaemia and hepatic glycogen and triglyceride overaccumulation. The latter leads to steatohepatitis, cirrhosis, and the formation of hepatic adenomas and carcinomas. Currently, little is known about the function of various organelles and their impact on metabolism in GSDIa. Accordingly, we investigated mitochondrial function in cell culture and mouse models of GSDIa. We found impairments in oxidative phosphorylation and changes in TCA cycle metabolites, as well as decreased mitochondrial membrane potential and deranged mitochondrial ultra-structure in these model systems. Mitochondrial content also was decreased, likely secondary to decreased mitochondrial biogenesis. These deleterious effects culminated in the activation of the mitochondrial apoptosis pathway. Taken together, our results demonstrate a role for mitochondrial dysfunction in the pathogenesis of GSDIa, and identify a new potential target for the treatment of this disease. They also provide new insight into the role of carbohydrate overload on mitochondrial function in other hepatic diseases, such as non-alcoholic fatty liver disease.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1. Mitochondrial respiration is impaired in G6PC KD versus control siRNA treated AML-12 cells.

G6PC was knocked-down in AML-12 cells for 96 hours, then mitochondrial oximetry analysis was performed using a Seahorse XF24 mitochondrial flux analyser. Basal respiration, ATP turnover, Maximal Respiration, and Spare Mitochondrial capacity were measured/calculated as described in the methods section. Oxygen consumption was normalised to total cellular protein content. For all parts, n = 6, error bars represent SEM, *represents p 

Figure 2. The tri-carboxylic acid cycle function is impaired in G6PC KO mice.

(A) Organic acid analysis in the livers G6PC KO mice (KO, n = 6) compared to wild-type (WT, n = 10) mice. (B) Analysis of amino acid levels in the same samples. (C) Schematic of the changes of the species in the TCA cycle. Species in green were increased in G6PC KO mouse livers relative to WT livers, while those in red were decreased. Species in blue showed no significant change between groups, while those in black were not directly measured. For all parts, error bars represent SEM, *represents p < 0.05.

Figure 3. Mitochondrial morphology is deranged in the livers of G6PC KO mice and G6PC KD AML-12 cells (96 hour knockdown).

Mitochondrial morphology was analysed in ultrathin sections of mouse liver (A) and AML-12 cells (B) by electron microscopy. Mitochondrial morphology in the wild-type mice and control cells were within normal limits, whereas in the KO mice and KD cells, the mitochondria were distended, and swollen, with effacement of the cristae, disruption of the mitochondrial membranes, and influx of cytoplasmic contents into the mitochondria. Representative images from the mouse liver (A) and KD cells (B) are displayed. Scale bars are marked on each image. Frames box areas shown at high magnification.

Figure 4. Mitochondrial content is reduced in GSDIa models.

(A) The protein levels of various key mitochondrial proteins are reduced following G6PC KD for 96 hours in AML-12 cells (n = 3). (B) The copy number of mitochondrial DNA is also reduced following G6PC KD for 96 hours in AML-12 cells (n = 6). (C) G6PC KO mouse livers also show decreased levels of mitochondrial proteins (n = 3). *Represents p < 0.05, error bars represent SEM.

Figure 5. Key factors involved in mitochondrial biogenesis are reduced following G6PC KD for 96 hours in AML-12 cells.

(A) Analysis of mRNA expression of genes involved in mitochondrial biogenesis, quality control and mitophagy shows a decrease in expression of genes related to mitochondrial biogenesis, with no change in expression of key fission and fusion genes. (n = 3). (B) Protein levels of TFAM are reduced following G6PC KD, however those of NRF1 show no significant change (n = 5). *Represents p < 0.05, error bars represent SEM.

Figure 6. Protein levels of key transcription factors involved in mitochondrial biogenesis.

(A) PGC1α levels are reduced in G6PC KD AML-12 cells (n = 5). (B) PGC1α levels are also reduced in G6PC KO mouse livers (n = 3). (C) Levels of ERRα are unchanged in G6PC KO mouse livers as compared to WT livers (n = 3). *Represents p < 0.05, error bars represent SEM.

Figure 7. The mitochondrial apoptosis pathway is up-regulated in GSD1a.

(A) Mitochondrial membrane potential is reduced in G6PC KD AML-12 cells 72 hours after knockdown. (B) Cytosolic cytochrome c is increased in G6PC KD AML-12 cells. (C) Caspase 9 and caspase 3 cleavage is increased in G6PC KD AML-12 cells. (D,E) Caspase 9 (D) and caspase 3 (E) cleavage is increasted in G6PC KO mice. For all parts except A, n = 3, error bars represent SEM, and *represents p < 0.05. For A, n = 6.