Roberta Resaz

Background: Mutations in SLC37A4, which encodes the intracellular glucose transporter G6PT, cause the rare glycogen storage disease type 1b (GSD1b). A long-term consequence of GSD1b is kidney failure, which requires kidney replacement therapy. The main protein markers of proximal tubule function, including NaPi2A, NHE3, SGLT2, GLUT2, and AQP1, are downregulated as part of the disease phenotype. Methods: We utilized an inducible mouse model of GSD1b, TM-G6PT^-/-, to show that glycogen accumulation plays a crucial role in altering proximal tubule morphology and function. To limit glucose entry into proximal tubule cells and, thus, to prevent glycogen accumulation, we administered a SGLT2-inhibitor, dapagliflozin, to TM-G6PT^-/- mice. Results: In proximal tubule cells, G6PT suppression stimulates the upregulation and activity of hexokinase I, which increases availability of the reabsorbed glucose for intracellular metabolism. Dapagliflozin prevented glycogen accumulation and improved kidney morphology by promoting a metabolic switch from glycogen synthesis towards lysis and restored expression levels of the main proximal tubule functional markers. Conclusion: We provide proof of concept for the efficacy of dapagliflozin in preserving kidney function in GSD1b mice. Our findings could represent the basis for repurposing this drug to treat GSD1b patients.

Glycogen storage disease type Ia (GSDIa) is an inherited metabolic disorder caused by mutations in the enzyme glucose-6-phosphatase-α (G6Pase-α). Affected individuals develop renal and liver complications, including the development of hepatocellular adenoma/carcinoma and kidney failure. The purpose of this study was to identify potential biomarkers of the evolution of the disease in GSDIa patients. To this end, we analyzed the expression of exosomal microRNAs (Exo-miRs) in the plasma exosomes of 45 patients aged 6 to 63 years. Plasma from age-matched normal individuals were used as controls. We found that the altered expression of several Exo-miRs correlates with the pathologic state of the patients and might help to monitor the progression of the disease and the development of late GSDIa-associated complications.

Glycogen storage disease type 1a (GSD-1a) is a rare genetic disease caused by mutations in the catalytic subunit of the enzyme glucose-6-phosphatase-alpha (G6Pase-α). The majority of patients develop long-term complications including renal failure and hepatocellular adenoma/carcinoma. The purpose of this study was to ascertain the proteomic changes in the liver of LS-G6pc-/- mice, a murine model of GSD-1a, in comparison with wild type mice to identify potential biomarkers of the pathophysiology of the affected liver. We used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to analyze liver lysates from a total of 20 LS-G6pc-/- and 18 wild type (WT) mice. We compared the proteomic expression profile of LS-G6pc-/- and WT mice. We identified 4138 significantly expressed proteins 1243 of which were differentially represented. Network and pathway analyses indicate that LS-G6pc-/- livers display an age-dependent modulation of the expression of proteins involved in specific biological processes associated with increased progression of liver disease. Moreover, we found upregulation of proteins involved in the process of tissue inflammation and macrophage polarization toward the M2 phenotype in LS-G6pc-/- mice with adenomas. Our results identify a metabolic reprogramming of glucose-6-P and a pathologic environment in the liver compatible with tumor development and progression.

Hepatocellular adenomas (HCAs) are benign tumors, of which the most serious complications are hemorrhage and malignant transformation to hepatocellular carcinoma (HCC). Among the various subtypes of HCA, the β-catenin-activated subtype (bHCA) is associated with greatest risk of malignant transformation. Magnetic resonance imaging (MRI) is an important tool to differentiate benign and malignant hepatic lesions, and preclinical experimental approaches may help to develop a method to identify MRI features associated with bHCA. HCAs are associated with various pathologies, including glycogen storage disease 1a (GSD1a). Here, we utilized a mouse model for GSD1a that develops HCA and HCC, and analyzed the mice in order to distinguish low-risk from high-risk tumors. Animals were scanned by MRI using a hepato-specific contrast agent. The mice were sacrificed after MRI and their lesions were classified using immunohistochemistry. We observed that 45% of the animals developed focal lesions, and MRI identified four different patterns after contrast administration: isointense, hyperintense and hypointense lesions, and lesions with peripheral contrast enhancement. After contrast administration, only bHCA and HCC were hypointense in T1-weighted imaging and mildly hyperintense in T2-weighted imaging. Thus, high-risk adenomas display MRI features clearly distinguishable from those exhibited by low-risk adenomas, indicating that MRI is a reliable method for early diagnosis and classification of HCA, necessary for correct patient management.

Background and aims: Glycogen storage disease type Ib (GSD1b) is a rare metabolic and immune disorder caused by a deficiency in the glucose‐6‐phosphate transporter (G6PT) and characterized by impaired glucose homeostasis, myeloid dysfunction, and long‐term risk of hepatocellular adenomas. Despite maximal therapy, based on a strict diet and on granulocyte colony‐stimulating factor treatment, long‐term severe complications still develop. Understanding the pathophysiology of GSD1b is a prerequisite to develop new therapeutic strategies and depends on the availability of animal models. The G6PT‐KO mouse mimics the human disease but is very fragile and rarely survives weaning. We generated a conditional G6PT‐deficient mouse as an alternative model for studying the long‐term pathophysiology of the disease. We utilized this conditional mouse to develop an inducible G6PT‐KO model to allow temporally regulated G6PT deletion by the administration of tamoxifen (TM).Methods: We generated a conditional G6PT‐deficient mouse utilizing the CRElox strategy. Histology, histochemistry, and phenotype analyses were performed at different times after TM‐induced G6PT inactivation. Neutrophils and monocytes were isolated and analyzed for functional activity with standard techniques.Results: The G6PT‐inducible KO mice display the expected disturbances of G6P metabolism and myeloid dysfunctions of the human disorder, even though with a milder intensity.Conclusions: TM‐induced inactivation of G6PT in these mice leads to a phenotype which mimics that of human GSD1b patients. The conditional mice we have generated represent an excellent tool to study the tissue‐specific role of the G6PT gene and the mechanism of long‐term complications in GSD1b.

Glycogen storage disease type 1a (GSD-1a) is caused by a deficiency in glucose-6-phosphatase-α (G6Pase-α), and is characterized by impaired glucose homeostasis and a high risk of developing hepatocellular adenomas (HCAs). A globally G6Pase-α-deficient (G6pc^−/−) mouse model that shows pathological features similar to those of humans with GSD-1a has been developed. These mice show a very severe phenotype of disturbed glucose homeostasis and rarely live beyond weaning. We generated liver-specific G6Pase-α-deficient (LS‑G6pc^−/−) mice as an alternative animal model for studying the long-term pathophysiology of the liver and the potential treatment strategies, such as cell therapy. LS‑G6pc^−/− mice were viable and exhibited normal glucose profiles in the fed state, but showed significantly lower blood glucose levels than their control littermates after 6 hours of fasting. LS‑G6pc^−/− mice developed hepatomegaly with glycogen accumulation and hepatic steatosis, and progressive hepatic degeneration. Ninety percent of the mice analyzed developed amyloidosis by 12 months of age. Finally, 25% of the mice sacrificed at age 10–20 months showed the presence of multiple HCAs and in one case late development of hepatocellular carcinoma (HCC). In conclusion, LS‑G6pc^−/− mice manifest hepatic symptoms similar to those of human GSD-1a and, therefore, represent a valid model to evaluate long-term liver pathogenesis of GSD-1a.

Dbl is the prototype of a large family of GDP–GTP exchange factors for small GTPases of the Rho family. In vitro, Dbl is known to activate Rho, Rac, and Cdc42 and to induce a transformed phenotype in murine fibroblasts. We previously reported that Dbl-null mice are viable and fertile but display defective dendrite elongation of distinct subpopulations of cortical neurons, suggesting a role of Dbl in controlling dendritic growth. To gain deeper insights into the role of Dbl in development and disease, we attempted a knock-in approach to create an endogenous allele that encodes a missense-mutation-mediated loss of function in the DH domain. We generated, by gene targeting technology, a mutant mouse strain by inserting a mutagenized human proto-Dbl cDNA clone expressing only the Dbl N terminus regulatory sequence at the starting codon of murine exon 1. Animals were monitored over a 21-month period, and necropsy specimens were collected for histological examination and immunohistochemistry analysis. Dbl knock-in mice are viable and did not manifest either decreased reproductive performances or gross developmental phenotype but revealed a reduced lifespan compared to wild-type (w.t.) mice and showed, with aging, a B cell lymphoproliferation that often has features of a frank diffuse large B cell lymphoma. Moreover, Dbl knock-in male mice displayed an increased incidence of lung adenoma compared to w.t. mice. These data indicate that Dbl is a tumor susceptibility gene in mice and that loss of function of Dbl DH domain by genetic missense mutations is responsible for induction of diffuse large B cell lymphoma.