Potential use of other starch sources in the treatment of glycogen storage disease type Ia - an in vitro study.

BACKGROUND: Glycogen storage disease type Ia (GSD-Ia) is one of the most common hepatic GSD. Its treatment mainly consists of a diet including a high intake of slow-digestion carbohydrates such as raw cornstarch and the restriction of simple sugars. This enables the maintenance of euglycemia and prevents secondary metabolic disorders. Starch is a glucose polymer formed by amylose and amylopectin, which can be obtained from distinct sources. Although uncooked cornstarch has been successfully used in the treatment of GSD-Ia, it can lead to hyperglycemia and weight gain. in vitro andin vivo tests indicated that sweet manioc starch can be potentially used in the treatment of GSD-Ia. RESULTS: The moisture analysis revealed a variation from 10.3 to 12.8% in the sweet manioc starch samples, whereas the moisture content of uncooked cornstarch ranged from 7.3 to 11.1%. Quantifiable sugar was detected in 3/5 samples of sweet manioc starch and 1/3 samples of uncooked cornstarch. Notably, this uncooked cornstarch brand is widely employed in GSD-Ia treatment in Brazil. Products B and E had higher values of amylopectin and undetectable levels of sugars. A clinical trial is warranted to compare samples F and G and determine the impact of sugar trace in the same dietary source of starch. CONCLUSIONS: Collectively, the results demonstrated possible therapeutic alternatives for GSD-Ia in addition to traditional uncooked cornstarch.

Gut Dysbiosis Drives Inflammatory Bowel Disease Through the CCL4L2-VSIR Axis in Glycogen Storage Disease.

Patients with glycogen storage disease type Ib (GSD-Ib) frequently have inflammatory bowel disease (IBD). however, the underlying etiology remains unclear. Herein, this study finds that digestive symptoms are commonly observed in patients with GSD-Ib, presenting as single or multiple scattered deep round ulcers, inflammatory pseudo-polyps, obstructions, and strictures, which differ substantially from those in typical IBD. Distinct microbiota profiling and single-cell clustering of colonic mucosae in patients with GSD are conducted. Heterogeneous oral pathogenic enteric outgrowth induced by GSD is a potent inducer of gut microbiota immaturity and colonic macrophage accumulation. Specifically, a unique population of macrophages with high CCL4L2 expression is identified in response to pathogenic bacteria in the intestine. Hyper-activation of the CCL4L2-VSIR axis leads to increased expression of AGR2 and ZG16 in epithelial cells, which mediates the unique progression of IBD in GSD-Ib. Collectively, the microbiota-driven pathomechanism of IBD is demonstrated in GSD-Ib and revealed the active role of the CCL4L2-VSIR axis in the interaction between the microbiota and colonic mucosal immunity. Thus, targeting gut dysbiosis and/or the CCL4L2-VISR axis may represent a potential therapy for GSD-associated IBD.

Endogenous Glucose Production in Patients With Glycogen Storage Disease Type Ia Estimated by Oral D-[6,6-2H2]-glucose.

CONTEXT: Glycogen storage disease type Ia (GSDIa) is an inborn metabolic disorder characterized by impaired endogenous glucose production (EGP). Monitoring of patients with GSDIa is prioritized because of ongoing treatment developments. Stable isotope tracers may enable reliable EGP monitoring. OBJECTIVE: The aim of this study was to prospectively assess the rate of appearance of endogenous glucose into the bloodstream (Ra) in patients with GSDIa after a single oral D-[6,6-2H2]-glucose dose. METHODS: Ten adult patients with GSDIa and 10 age-, sex-, and body mass index-matched healthy volunteers (HVs) were enrolled. For each participant, 3 oral glucose tracer tests were performed: (1) preprandial/fasted, (2) postprandial, and (3) randomly fed states. Dried blood spots were collected before D-[6,6-2H2]-glucose administration and 10, 20, 30, 40, 50, 60, 75, 90, and 120 minutes thereafter. RESULTS: Glucose Ra in fasted HVs was consistent with previously reported data. The time-averaged glucose Ra was significantly higher in (1) preprandial/fasted patients with GSDIa than HV and (2) postprandial HV compared with fasted HV(P < .05). A progressive decrease in glucose Ra was observed in preprandial/fasted patients with GSDIa; the change in glucose Ra time-course was directly correlated with the change in capillary glucose (P < .05). CONCLUSION: This is the first study to quantify glucose Ra in patients with GSDIa using oral D-[6,6-2H2] glucose. The test can reliably estimate EGP under conditions in which fasting tolerance is unaffected but does not discriminate between relative contributions of EGP (eg, liver, kidney) and exogenous sources (eg, dietary cornstarch). Future application is warranted for longitudinal monitoring after novel genome based treatments in patients with GSDIa in whom nocturnal dietary management can be discontinued.

Mutational analysis and clinical investigations of medically diagnosed GSD 1a patients from Pakistan.

Glycogen storage disease type I (GSD I) is a rare autosomal recessive inborn error of carbohydrate metabolism caused by the defects of glucose-6-phosphatase complex (G6PC). Disease causing variants in the G6PC gene, located on chromosome 17q21 result in glycogen storage disease type Ia (GSD Ia). Age of onset of GSD Ia ranges from 0.5 to 25 years with presenting features including hemorrhage, hepatic, physical and blood related abnormalities. The overall goal of proposed study was clinical and genetic characterization of GSD Ia cases from Pakistani population. This study included forty GSD Ia cases presenting with heterogeneous clinical profile including hypoglycemia, hepatomegaly, lactic acidosis i.e., pH less than 7.2, hyperuricemia, seizures, epistaxis, hypertriglyceridemia (more than180 mg/dl) and sometimes short stature. All coding exons and intron-exon boundaries of G6PC gene were screened to identify pathogenic variant in 20 patients based on availability of DNA samples and willingness to participate in molecular analysis. Pathogenic variant analysis was done using PCR-Sanger sequencing method and pathogenic effect predictions for identified variants were carried out using PROVEAN, MutationTaster, Polyphen 2, HOPE, Varsome, CADD, DANN, SIFT and HSF software. Overall, 21 variants were detected including 8 novel disease causing variants i.e., G6PC (NM_000151.4):c.71A>C (p.Gln24Pro), c.109G>C(p.Ala37Pro), c.133G>C(p.Val45Leu), c.49_50insT c.205G>A(p.Asp69Asn), c.244C>A(p.Gln82Lys) c.322A>C(p.Thr108Pro) and c.322A>C(p.Cys284Tyr) in the screened regions of G6PC gene. Out of 13 identified polymorphisms, 3 were identified in heterozygous condition while 10 were found in homozygous condition. This study revealed clinical presentation of GSD Ia cases from Pakistan and identification of novel disease-causing sequence variants in coding region and intron-exon boundaries of G6PC gene.

Aberrant glucose metabolism underlies impaired macrophage differentiation in glycogen storage disease type Ib.

Glycogen storage disease type Ib (GSD-Ib) is an autosomal recessive disorder caused by a deficiency in the glucose-6-phosphate (G6P) transporter (G6PT) that is responsible for transporting G6P into the endoplasmic reticulum. GSD-Ib is characterized by disturbances in glucose homeostasis, neutropenia, and neutrophil dysfunction. Although some studies have explored neutrophils abnormalities in GSD-Ib, investigations regarding monocytes/macrophages remain limited so far. In this study, we examined the impact of G6PT deficiency on monocyte-to-macrophage differentiation using bone marrow-derived monocytes from G6pt-/- mice as well as G6PT-deficient human THP-1 monocytes. Our findings revealed that G6PT-deficient monocytes exhibited immature differentiation into macrophages. Notably, the impaired differentiation observed in G6PT-deficient monocytes seemed to be associated with abnormal glucose metabolism, characterized by enhanced glucose consumption through glycolysis, even under quiescent conditions with oxidative phosphorylation. Furthermore, we observed a reduced secretion of inflammatory cytokines in G6PT-deficient THP-1 monocytes during the inflammatory response, despite their elevated glucose consumption. In conclusion, this study sheds light on the significance of G6PT in monocyte-to-macrophage differentiation and underscores its importance in maintaining glucose homeostasis and supporting immune response in GSD-Ib. These findings may contribute to a better understanding of the pathogenesis of GSD-Ib and potentially pave the way for the development of targeted therapeutic interventions.

CRISPR/Cas9-based double-strand oligonucleotide insertion strategy corrects metabolic abnormalities in murine glycogen storage disease type-Ia.

Glycogen storage disease type-Ia (GSD-Ia), characterized by impaired blood glucose homeostasis, is caused by a deficiency in glucose-6-phosphatase-α (G6Pase-α or G6PC). Using the G6pc-R83C mouse model of GSD-Ia, we explored a CRISPR/Cas9-based double-strand DNA oligonucleotide (dsODN) insertional strategy that uses the nonhomologous end-joining repair mechanism to correct the pathogenic p.R83C variant in G6pc exon-2. The strategy is based on the insertion of a short dsODN into G6pc exon-2 to disrupt the native exon and to introduce an additional splice acceptor site and the correcting sequence. When transcribed and spliced, the edited gene would generate a wild-type mRNA encoding the native G6Pase-α protein. The editing reagents formulated in lipid nanoparticles (LNPs) were delivered to the liver. Mice were treated either with one dose of LNP-dsODN at age 4 weeks or with two doses of LNP-dsODN at age 2 and 4 weeks. The G6pc-R83C mice receiving successful editing expressed ~4% of normal hepatic G6Pase-α activity, maintained glucose homeostasis, lacked hypoglycemic seizures, and displayed normalized blood metabolite profile. The outcomes are consistent with preclinical studies supporting previous gene augmentation therapy which is currently in clinical trials. This editing strategy may offer the basis for a therapeutic approach with an earlier clinical intervention than gene augmentation, with the additional benefit of a potentially permanent correction of the GSD-Ia phenotype.

Current understanding on pathogenesis and effective treatment of glycogen storage disease type Ib with empagliflozin: new insights coming from diabetes for its potential implications in other metabolic disorders.

Glycogen storage type Ib (GSDIb) is a rare inborn error of metabolism caused by glucose-6-phosphate transporter (G6PT, SLC37A4) deficiency. G6PT defect results in excessive accumulation of glycogen and fat in the liver, kidney, and intestinal mucosa and into both glycogenolysis and gluconeogenesis impairment. Clinical features include hepatomegaly, hypoglycemia, lactic acidemia, hyperuricemia, hyperlipidemia, and growth retardation. Long-term complications are liver adenoma, hepatocarcinoma, nephropathy and osteoporosis. The hallmark of GSDIb is neutropenia, with impaired neutrophil function, recurrent infections and inflammatory bowel disease. Alongside classical nutritional therapy with carbohydrates supplementation and immunological therapy with granulocyte colony-stimulating factor, the emerging role of 1,5-anhydroglucitol in the pathogenesis of neutrophil dysfunction led to repurpose empagliflozin, an inhibitor of the renal glucose transporter SGLT2: the current literature of its off-label use in GSDIb patients reports beneficial effects on neutrophil dysfunction and its clinical consequences. Surprisingly, this glucose-lowering drug ameliorated the glycemic and metabolic control in GSDIb patients. Furthermore, numerous studies from big cohorts of type 2 diabetes patients showed the efficacy of empagliflozin in reducing the cardiovascular risk, the progression of kidney disease, the NAFLD and the metabolic syndrome. Beneficial effects have also been described on peripheral neuropathy in a prediabetic rat model. Increasing evidences highlight the role of empagliflozin in regulating the cellular energy sensors SIRT1/AMPK and Akt/mTOR, which leads to improvement of mitochondrial structure and function, stimulation of autophagy, decrease of oxidative stress and suppression of inflammation. Modulation of these pathways shift the oxidative metabolism from carbohydrates to lipids oxidation and results crucial in reducing insulin levels, insulin resistance, glucotoxicity and lipotoxicity. For its pleiotropic effects, empagliflozin appears to be a good candidate for drug repurposing also in other metabolic diseases presenting with hypoglycemia, organ damage, mitochondrial dysfunction and defective autophagy.

Amnio acid substitution at position 298 of human glucose-6 phosphatase-α significantly impacts its stability in mammalian cells.

Glucose-6-phosphatase-α (G6Pase-α) catalyzes the hydrolysis of glucose-6-phosphate to glucose and functions as a key regulator in maintaining blood glucose homeostasis. Deficiency in G6Pase-α causes glycogen storage disease 1a (GSD1a), an inherited disorder characterized by life-threatening hypoglycemia and other long-term complications. We have developed a potential mRNA-based therapy for GSD1a and demonstrated that a human G6Pase-α (hG6Pase-α) variant harboring a single serine (S) to cysteine (C) substitution at the amino acid site 298 (S298C) had > twofold increase in protein expression, resulting in improved in vivo efficacy. Here, we sought to investigate the mechanisms contributing to the increased expression of the S298C variant. Mutagenesis of hG6Pase-α identified distinct protein variants at the 298 amino acid position with substantial reduction in protein expression in cultured cells. Kinetic analysis of expression and subcellular localization in mammalian cells, combined with cell-free in vitro translation assays, revealed that altered protein expression stemmed from differences in cellular protein stability rather than biosynthetic rates. Site-specific mutagenesis studies targeting other cysteines of the hG6Pase-α S298C variant suggest the observed improvements in stability are not due to additional disulfide bond formation. The glycosylation at Asparagine (N)-96 is critical in maintaining enzymatic activity and mutations at position 298 mainly affected glycosylated forms of hG6Pase-α. Finally, proteasome inhibition by lactacystin improved expression levels of unstable hG6Pase-α variants. Taken together, these data uncover a critical role for a single amino acid substitution impacting the stability of G6Pase-α and provide insights into the molecular genetics of GSD1a and protein engineering for therapeutic development.

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.

Studies on glycogen storage disease type 1a animal models: a brief perspective.

Glycogen storage disease type 1 (GSD1) is a rare hereditary monogenic disease characterized by the disturbed glucose metabolism. The most widespread variant of GSD1 is GSD1a, which is a deficiency of glucose-6-phosphatase-ɑ. Glucose-6-phosphatase-ɑ is expressed only in liver, kidney, and intestine, and these organs are primarily affected by its deficiency, and long-term complications of GSD1a include hepatic tumors and chronic liver disease. This article is a brief overview of existing animal models for GSD1a, from the first mouse model of 1996 to modern CRISPR/Cas9-generated ones. First whole-body murine models demonstrated exact metabolic symptoms of GSD1a, but the animals did not survive weaning. The protocol for glucose treatment allowed prolonged survival of affected animals, but long-term complications, such as hepatic tumorigenesis, could not be investigated. Next, organ-specific knockout models were developed, and most of the metabolic research was performed on liver glucose-6-phosphate-deficient mice. Naturally occuring mutation was also discovered in dogs. All these models are widely used to study GSD1a from metabolic and physiological standpoints and to develop possible treatments involving gene therapy. Research performed using these models helped elucidate the role of glycogen and lipid accumulation, hypoxia, mitochondrial dysfunction, and autophagy impairment in long-term complications of GSD1a, including hepatic tumorigenesis. Recently, gene replacement therapy and genome editing were tested on described models, and some of the developed approaches have reached clinical trials.

Dapagliflozin Prevents Kidney Glycogen Accumulation and Improves Renal Proximal Tubule Cell Functions in a Mouse Model of Glycogen Storage Disease Type 1b.

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 KRT. 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 an 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 toward lysis and by restoring 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 patients with GSD1b.

Glycogen storage diseases with liver involvement: a literature review of GSD type 0, IV, VI, IX and XI.

BACKGROUND: Glycogen storage diseases (GSDs) with liver involvement are classified into types 0, I, III, IV, VI, IX and XI, depending on the affected enzyme. Hypoglycemia and hepatomegaly are hallmarks of disease, but muscular and renal tubular involvement, dyslipidemia and osteopenia can develop. Considering the paucity of literature available, herein we provide a narrative review of these latter forms of GSDs. MAIN BODY: Diagnosis is based on clinical manifestations and laboratory test results, but molecular analysis is often necessary to distinguish the various forms, whose presentation can be similar. Compared to GSD type I and III, which are characterized by a more severe impact on metabolic and glycemic homeostasis, GSD type 0, VI, IX and XI are usually known to be responsive to the nutritional treatment for achieving a balanced metabolic homeostasis in the pediatric age. However, some patients can exhibit a more severe phenotype and an important progression of the liver and muscular disease. The effects of dietary adjustments in GSD type IV are encouraging, but data are limited. CONCLUSIONS: Early diagnosis allows a good metabolic control, with improvement of quality of life and prognosis, therefore we underline the importance of building a proper knowledge among physicians about these rare conditions. Regular monitoring is necessary to restrain disease progression and complications.

Molecular mechanisms of aberrant neutrophil differentiation in glycogen storage disease type Ib.

Glycogen storage disease type Ib (GSD-Ib), characterized by impaired glucose homeostasis, neutropenia, and neutrophil dysfunction, is caused by a deficiency in glucose-6-phosphate transporter (G6PT). Neutropenia in GSD-Ib has been known to result from enhanced apoptosis of neutrophils. However, it has also been raised that neutrophil maturation arrest in the bone marrow would contribute to neutropenia. We now show that G6pt-/- mice exhibit severe neutropenia and impaired neutrophil differentiation in the bone marrow. To investigate the role of G6PT in myeloid progenitor cells, the G6PT gene was mutated using CRISPR/Cas9 system, and single cell-derived G6PT-/- human promyelocyte HL-60 cell lines were established. The G6PT-/- HL-60s exhibited impaired neutrophil differentiation, which is associated with two mechanisms: (i) abnormal lipid metabolism causing a delayed metabolic reprogramming and (ii) reduced nuclear transcriptional activity of peroxisome proliferator-activated receptor-γ (PPARγ) in G6PT-/- HL-60s. In this study, we demonstrated that G6PT is essential for neutrophil differentiation of myeloid progenitor cells and regulates PPARγ activity.

Liver transplantation in glycogen storage disease: a single-center experience.

BACKGROUND: Glycogen storage diseases (GSDs) are inherited glycogen metabolic disorders which have various subtypes. GSDs of type I, III, IV, VI, and IX show liver involvement and are considered as hepatic types of GSDs. Thus, liver transplantation (LT) has been proposed as a final therapy for these types of GSD. LT corrects the primary hepatic enzyme defect; however, the long-term outcomes of LT in these patients have not been extensively evaluated so far. There are few reports in the English literature about the outcome of GSD patients after LT. There has been no report from Iran. The present retrospective study aimed to evaluate the long-term outcomes of eight patients with GSD types I, III, and IV who underwent LT in the affiliated hospitals of Shiraz University of Medical Sciences, from March 2013 to June 2021. During this period, there were no patients with GSD VI and IX identified in this center. RESULTS: The median time of diagnosis of the GSDs and at transplant was 1 year and 11 years, respectively. All eight transplanted patients were alive at the time of follow-up in this study. None of them required a re-transplant. All of the patients showed normalized liver enzymes after LT with no sign of hypoglycemia. CONCLUSIONS: LT is an achievable treatment for end-stage hepatic involvement of GSDs with a cure for metabolic deficiency. Our experience in these eight patients shows a favorable outcome with no mortality and no major complication.

Biophysical and functional properties of purified glucose-6-phosphatase catalytic subunit 1.

Glucose-6-phosphatase catalytic subunit 1 (G6PC1) plays a critical role in hepatic glucose production during fasting by mediating the terminal step of the gluconeogenesis and glycogenolysis pathways. In concert with accessory transport proteins, this membrane-integrated enzyme catalyzes glucose production from glucose-6-phosphate (G6P) to support blood glucose homeostasis. Consistent with its metabolic function, dysregulation of G6PC1 gene expression contributes to diabetes, and mutations that impair phosphohydrolase activity form the clinical basis of glycogen storage disease type 1a. Despite its relevance to health and disease, a comprehensive view of G6PC1 structure and mechanism has been limited by the absence of expression and purification strategies that isolate the enzyme in a functional form. In this report, we apply a suite of biophysical and biochemical tools to fingerprint the in vitro attributes of catalytically active G6PC1 solubilized in lauryl maltose neopentyl glycol (LMNG) detergent micelles. When purified from Sf9 insect cell membranes, the glycosylated mouse ortholog (mG6PC1) recapitulated functional properties observed previously in intact hepatic microsomes and displayed the highest specific activity reported to date. Additionally, our results establish a direct correlation between the catalytic and structural stability of mG6PC1, which is underscored by the enhanced thermostability conferred by phosphatidylcholine and the cholesterol analog cholesteryl hemisuccinate. In contrast, the N96A variant, which blocks N-linked glycosylation, reduced thermostability. The methodologies described here overcome long-standing obstacles in the field and lay the necessary groundwork for a detailed analysis of the mechanistic structural biology of G6PC1 and its role in complex metabolic disorders.

Hepatocyte-specific glucose-6-phosphatase deficiency disturbs platelet aggregation and decreases blood monocytes upon fasting-induced hypoglycemia.

OBJECTIVE: Glycogen storage disease type 1a (GSD Ia) is a rare inherited metabolic disorder caused by mutations in the glucose-6-phosphatase (G6PC1) gene. When untreated, GSD Ia leads to severe fasting-induced hypoglycemia. Although current intensive dietary management aims to prevent hypoglycemia, patients still experience hypoglycemic events. Poor glycemic control in GSD Ia is associated with hypertriglyceridemia, hepatocellular adenoma and carcinoma, and also with an increased bleeding tendency of unknown origin. METHODS: To evaluate the effect of glycemic control on leukocyte levels and coagulation in GSD Ia, we employed hepatocyte-specific G6pc1 deficient (L-G6pc-/-) mice under fed or fasted conditions, to match good or poor glycemic control in GSD Ia, respectively. RESULTS: We found that fasting-induced hypoglycemia in L-G6pc-/- mice decreased blood leukocytes, specifically proinflammatory Ly6Chi monocytes, compared to controls. Refeeding reversed this decrease. The decrease in Ly6Chi monocytes was accompanied by an increase in plasma corticosterone levels and was prevented by the glucocorticoid receptor antagonist mifepristone. Further, fasting-induced hypoglycemia in L-G6pc-/- mice prolonged bleeding time in the tail vein bleeding assay, with reversal by refeeding. This could not be explained by changes in coagulation factors V, VII, or VIII, or von Willebrand factor. While the prothrombin and activated partial thromboplastin time as well as total platelet counts were not affected by fasting-induced hypoglycemia in L-G6pc-/- mice, ADP-induced platelet aggregation was disturbed. CONCLUSIONS: These studies reveal a relationship between fasting-induced hypoglycemia, decreased blood monocytes, and disturbed platelet aggregation in L-G6pc-/- mice. While disturbed platelet aggregation likely accounts for the bleeding phenotype in GSD Ia, elevated plasma corticosterone decreases the levels of proinflammatory monocytes. These studies highlight the necessity of maintaining good glycemic control in GSD Ia.

A generic emergency protocol for patients with inborn errors of metabolism causing fasting intolerance: A retrospective, single-center study and the generation of www.emergencyprotocol.net.

Patients with inborn errors of metabolism causing fasting intolerance can experience acute metabolic decompensations. Long-term data on outcomes using emergency letters are lacking. This is a retrospective, observational, single-center study of the use of emergency letters based on a generic emergency protocol in patients with hepatic glycogen storage diseases (GSD) or fatty acid oxidation disorders (FAOD). Data on hospital admissions, initial laboratory results, and serious adverse events were collected. Subsequently, the website www.emergencyprotocol.net was generated in the context of the CONNECT MetabERN eHealth project following multiple meetings, protocol revisions, and translations. Representing 470 emergency protocol years, 127 hospital admissions were documented in 54/128 (42%) patients who made use of emergency letters generated based on the generic emergency protocol. Hypoglycemia (here defined as glucose concentration < 3.9 mmol/L) was reported in only 15% of hospital admissions and was uncommon in patients with ketotic GSD and patients with FAOD aged >5 years. Convulsions, coma, or death was not documented. By providing basic information, emergency letters for individual patients with hepatic GSD or the main FAOD can be generated at www.emergencyprotocol.net, in nine different languages. Generic emergency protocols are safe and easy for home management by the caregivers and the first hour in-hospital management to prevent metabolic emergencies in patients with hepatic GSD and medium-chain Acyl CoA dehydrogenase deficiency. The website www.emergencyprotocol.net is designed to support families and healthcare providers to generate personalized emergency letters for patients with hepatic GSD and the main FAOD.

Impaired Very-Low-Density Lipoprotein catabolism links hypoglycemia to hypertriglyceridemia in Glycogen Storage Disease type Ia.

Prevention of hypertriglyceridemia is one of the biomedical targets in Glycogen Storage Disease type Ia (GSD Ia) patients, yet it is unclear how hypoglycemia links to plasma triglyceride (TG) levels. We analyzed whole-body TG metabolism in normoglycemic (fed) and hypoglycemic (fasted) hepatocyte-specific glucose-6-phosphatase deficient (L-G6pc-/- ) mice. De novo fatty acid synthesis contributed substantially to hepatic TG accumulation in normoglycemic L-G6pc-/- mice. In hypoglycemic conditions, enhanced adipose tissue lipolysis was the main driver of liver steatosis, supported by elevated free fatty acid concentrations in GSD Ia mice and GSD Ia patients. Plasma very-low-density lipoprotein (VLDL) levels were increased in GSD Ia patients and in normoglycemic L-G6pc-/- mice, and further elevated in hypoglycemic L-G6pc-/- mice. VLDL-TG secretion rates were doubled in normo- and hypoglycemic L-G6pc-/- mice, while VLDL-TG catabolism was selectively inhibited in hypoglycemic L-G6pc-/- mice. In conclusion, fasting-induced hypoglycemia in L-G6pc-/- mice promotes adipose tissue lipolysis and arrests VLDL catabolism. This mechanism likely contributes to aggravated liver steatosis and dyslipidemia in GSD Ia patients with poor glycemic control and may explain clinical heterogeneity in hypertriglyceridemia between GSD Ia patients.

ANTHROPOMETRIC AND DIETARY ASSESSMENT OF PATIENTS WITH GLYCOGENOSIS TYPE I.

OBJECTIVE: To perform anthropometric and dietary evaluation of patients with glycogenosis type Ia and Ib. METHODS: This cross-sectional study is composed of a sample of 11 patients with glycogenosis divided into two subgroups according to the classification of glycogenosis (type Ia=5 and type Ib=6), aged between 4 and 20 years. The analyzed anthropometric variables were weight, height, body mass index, and measures of lean and fat body mass, which were compared with reference values. For dietary assessment, a food frequency questionnaire was used to calculate energy and macronutrients intake as well as the amount of raw cornstarch consumed. Mann-Whitney U test and Fisher's exact test were performed, considering a significance level of 5%. RESULTS: Patients ingested raw cornstarch in the amount of 0.49 to 1.34 g/kg/dose at a frequency of six times a day, which is lower than recommended (1.75-2.50 g/kg/dose, four times a day). The amount of energy intake was, on average, 50% higher than energy requirements; however, carbohydrate intake was below the adequacy percentage in 5/11 patients. Short stature was found in 4/10 patients; obesity, in 3/11; and muscle mass deficit, in 7/11. There were no statistical differences between the subgroups. CONCLUSIONS: In patients with glycogenosis type I, there was deficit in growth and muscle mass, but no differences were found between the subgroups (Ia and Ib). Although the diet did not exceed the adequacy of carbohydrates, about 1/3 of the patients presented obesity, probably due to higher energy intake.

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.