Identification of Hypoglycemic Glycolipids from Ipomoea murucoides by Affinity-Directed Fractionation, In Vitro, In Silico and Dynamic Light Scattering Analysis.

In the pursuit of identifying the novel resin glycoside modulators glucose-6-phosphatase and α-glucosidase enzymes, associated with blood sugar regulation, methanol-soluble extracts from the flowers of Ipomoea murucoides (cazahuate, Nahuatl), renowned for its abundance of glycolipids, were employed. The methanol-soluble extracts were fractionated by applying the affinity-directed method with glucose-6-phosphatase enzymes from a rat's liver and α-glucosidase enzymes from its intestines. Mass spectrometry and nuclear magnetic resonance were employed to identify the high-affinity compound as a free ligand following the release from the enzymatic complex. Gel permeation through a spin size-exclusion column allowed the separated high-affinity molecules to bind to glucose-6-phosphatase and α-glucosidase enzymes in solution, which led to the identification of some previously reported resin glycosides in the flowers of cazahuate, where a glycolipid mainly structurally related to murucoidin XIV was observed. In vitro studies demonstrated the modulating properties of resin glycosides on the glucose-6-phosphatase enzyme. Dynamic light scattering revealed conformational variations induced by resin glycosides on α-glucosidase enzyme, causing them to become more compact, akin to observations with the positive control, acarbose. These findings suggest that resin glycosides may serve as a potential source for phytotherapeutic agents with antihyperglycemic properties.

Biochemical and metabolic characterization of a G6PC2 inhibitor.

Three glucose-6-phosphatase catalytic subunits, that hydrolyze glucose-6-phosphate (G6P) to glucose and inorganic phosphate, have been identified, designated G6PC1-3, but only G6PC1 and G6PC2 have been implicated in the regulation of fasting blood glucose (FBG). Elevated FBG has been associated with multiple adverse clinical outcomes, including increased risk for type 2 diabetes and various cancers. Therefore, G6PC1 and G6PC2 inhibitors that lower FBG may be of prophylactic value for the prevention of multiple conditions. The studies described here characterize a G6PC2 inhibitor, designated VU0945627, previously identified as Compound 3. We show that VU0945627 preferentially inhibits human G6PC2 versus human G6PC1 but activates human G6PC3. VU0945627 is a mixed G6PC2 inhibitor, increasing the Km but reducing the Vmax for G6P hydrolysis. PyRx virtual docking to an AlphaFold2-derived G6PC2 structural model suggests VU0945627 binds two sites in human G6PC2. Mutation of residues in these sites reduces the inhibitory effect of VU0945627. VU0945627 does not inhibit mouse G6PC2 despite its 84% sequence identity with human G6PC2. Mutagenesis studies suggest this lack of inhibition of mouse G6PC2 is due, in part, to a change in residue 318 from histidine in human G6PC2 to proline in mouse G6PC2. Surprisingly, VU0945627 still inhibited glucose cycling in the mouse islet-derived ßTC-3 cell line. Studies using intact mouse liver microsomes and PyRx docking suggest that this observation can be explained by an ability of VU0945627 to also inhibit the G6P transporter SLC37A4. These data will inform future computational modeling studies designed to identify G6PC isoform-specific inhibitors.

Transcellular Barriers to Glucose Delivery in the Body.

Glucose is the universal fuel of most mammalian cells, and it is largely replenished through dietary intake. Glucose availability to tissues is paramount for the maintenance of homeostatic energetics and, hence, supply should match demand by the consuming organs. In its journey through the body, glucose encounters cellular barriers for transit at the levels of the absorbing intestinal epithelial wall, the renal epithelium mediating glucose reabsorption, and the tight capillary endothelia (especially in the brain). Glucose transiting through these cellular barriers must escape degradation to ensure optimal glucose delivery to the bloodstream or tissues. The liver, which stores glycogen and generates glucose de novo, must similarly be able to release it intact to the circulation. We present the most up-to-date knowledge on glucose handling by the gut, liver, brain endothelium, and kidney, and discuss underlying molecular mechanisms and open questions. Diseases associated with defects in glucose delivery and homeostasis are also briefly addressed. We propose that the universal problem of sparing glucose from catabolism in favor of translocation across the barriers posed by epithelia and endothelia is resolved through common mechanisms involving glucose transfer to the endoplasmic reticulum, from where glucose exits the cells via unconventional cellular mechanisms.

Identification of structural motifs critical for human G6PC2 function informed by sequence analysis and an AlphaFold2-predicted model.

G6PC2 encodes a glucose-6-phosphatase (G6Pase) catalytic subunit, primarily expressed in pancreatic islet ß cells, which modulates the sensitivity of insulin secretion to glucose and thereby regulates fasting blood glucose (FBG). Mutational analyses were conducted to validate an AlphaFold2 (AF2)-predicted structure of human G6PC2 in conjunction with a novel method to solubilize and purify human G6PC2 from a heterologous expression system. These analyses show that residues forming a predicted intramolecular disulfide bond are essential for G6PC2 expression and that residues forming part of a type 2 phosphatidic acid phosphatase (PAP2) motif are critical for enzyme activity. Additional mutagenesis shows that residues forming a predicted substrate cavity modulate enzyme activity and substrate specificity and residues forming a putative cholesterol recognition amino acid consensus (CRAC) motif influence protein expression or enzyme activity. This CRAC motif begins at residue 219, the site of a common G6PC2 non-synonymous single-nucleotide polymorphism (SNP), rs492594 (Val219Leu), though the functional impact of this SNP is disputed. In microsomal membrane preparations, the L219 variant has greater activity than the V219 variant, but this difference disappears when G6PC2 is purified in detergent micelles. We hypothesize that this was due to a differential association of the two variants with cholesterol. This concept was supported by the observation that the addition of cholesteryl hemi-succinate to the purified enzymes decreased the Vmax of the V219 and L219 variants ∼8-fold and ∼3 fold, respectively. We anticipate that these observations should support the rational development of G6PC2 inhibitors designed to lower FBG.

Inhibition of Wnt/ß-catenin signaling reduces renal fibrosis in murine glycogen storage disease type Ia.

Glycogen storage disease type Ia (GSD-Ia) is caused by a deficiency in the enzyme glucose-6-phosphatase-α (G6Pase-α or G6PC) that is expressed primarily in the gluconeogenic organs, namely liver, kidney cortex, and intestine. Renal G6Pase-α deficiency in GSD-Ia is characterized by impaired gluconeogenesis, nephromegaly due to elevated glycogen accumulation, and nephropathy caused, in part, by renal fibrosis, mediated by activation of the renin-angiotensin system (RAS). The Wnt/ß-catenin signaling regulates the expression of a variety of downstream mediators implicated in renal fibrosis, including multiple genes in the RAS. Sustained activation of Wnt/ß-catenin signaling is associated with the development and progression of renal fibrotic lesions that can lead to chronic kidney disease. In this study, we examined the molecular mechanism underlying GSD-Ia nephropathy. Damage to the kidney proximal tubules is known to trigger acute kidney injury (AKI) that can, in turn, activate Wnt/ß-catenin signaling. We show that GSD-Ia mice have AKI that leads to activation of the Wnt/ß-catenin/RAS axis. Renal fibrosis was demonstrated by increased renal levels of Snail1, α-smooth muscle actin (α-SMA), and extracellular matrix proteins, including collagen-Iα1 and collagen-IV. Treating GSD-Ia mice with a CBP/ß-catenin inhibitor, ICG-001, significantly decreased nuclear translocated active ß-catenin and reduced renal levels of renin, Snail1, α-SMA, and collagen-IV. The results suggest that inhibition of Wnt/ß-catenin signaling may be a promising therapeutic strategy for GSD-Ia nephropathy.

Intestinal gluconeogenesis: A translator of nutritional information needed for glycemic and emotional balance.

At the interface between the outside world and the self, the intestine is the first organ receiving nutritional information. One intestinal function, gluconeogenesis, is activated by various nutrients, particularly diets enriched in fiber or protein, and thus results in glucose production in the portal vein in the post-absorptive period. The detection of portal glucose induces a nervous signal controlling the activity of the central nuclei involved in the regulation of metabolism and emotional behavior. Induction of intestinal gluconeogenesis is necessary for the beneficial effects of fiber or protein-enriched diets on metabolism and emotional behavior. Through its ability to translate nutritional information from the diet to the brain's regulatory centers, intestinal gluconeogenesis plays an essential role in maintaining physiological balance.

Severe Hemolytic Anemia After Mothball Ingestion Complicated by an Undiagnosed Enzyme Deficiency: A Case Report.

Ingestion of mothballs containing naphthalene or paradichlorobenzene is known to cause hemolysis and methemoglobinemia secondary to severe oxidative stress, affecting the oxygen delivery system of the body. The gradual accumulation of oxidizing radicals in the setting of restricted glutathione availability leads to the oxidization of hemoglobin and other body proteins, ultimately causing cell destruction. In the setting of glucose-6-phosphate dehydrogenase deficiency (G6PDD), more pronounced symptoms and poor prognosis are anticipated as adequate nicotinamide adenine dinucleotide phosphate is not generated to protect red blood cells from oxidative injury, potentiating the hemolytic process further. Here, we report the case of a young male with mothball ingestion whose presentation and management were complicated by underlying undiagnosed G6PDD.

A molecular signature for the G6PC3/SLC37A2/SLC37A4 interactors in glioblastoma disease progression and in the acquisition of a brain cancer stem cell phenotype.

Background: Glycogen plays an important role in glucose homeostasis and contributes to key functions related to brain cancer cell survival in glioblastoma multiforme (GBM) disease progression. Such adaptive molecular mechanism is dependent on the glycogenolytic pathway and intracellular glucose-6-phosphate (G6P) sensing by brain cancer cells residing within those highly hypoxic tumors. The involvement of components of the glucose-6-phosphatase (G6Pase) system remains however elusive. Objective: We questioned the gene expression levels of components of the G6Pase system in GBM tissues and their functional impact in the control of the invasive and brain cancer stem cells (CSC) phenotypes. Methods: In silico analysis of transcript levels in GBM tumor tissues was done by GEPIA. Total RNA was extracted and gene expression of G6PC1-3 as well as of SLC37A1-4 members analyzed by qPCR in four human brain cancer cell lines and from clinically annotated brain tumor cDNA arrays. Transient siRNA-mediated gene silencing was used to assess the impact of TGF-ß-induced epithelial-to-mesenchymal transition (EMT) and cell chemotaxis. Three-dimensional (3D) neurosphere cultures were generated to recapitulate the brain CSC phenotype. Results: Higher expression in G6PC3, SLC37A2, and SLC37A4 was found in GBM tumor tissues in comparison to low-grade glioma and healthy tissue. The expression of these genes was also found elevated in established human U87, U251, U118, and U138 GBM cell models compared to human HepG2 hepatoma cells. SLC37A4/G6PC3, but not SLC37A2, levels were induced in 3D CD133/SOX2-positive U87 neurospheres when compared to 2D monolayers. Silencing of SLC37A4/G6PC3 altered TGF-ß-induced EMT biomarker SNAIL and cell chemotaxis. Conclusion: Two members of the G6Pase system, G6PC3 and SLC37A4, associate with GBM disease progression and regulate the metabolic reprogramming of an invasive and CSC phenotype. Such molecular signature may support their role in cancer cell survival and chemoresistance and become future therapeutic targets.

G6PC1 and G6PC2 influence G6P flux but not HSD11B1 activity.

In the endoplasmic reticulum (ER) lumen, glucose-6-phosphatase catalytic subunit 1 and 2 (G6PC1; G6PC2) hydrolyze glucose-6-phosphate (G6P) to glucose and inorganic phosphate whereas hexose-6-phosphate dehydrogenase (H6PD) hydrolyzes G6P to 6-phosphogluconate (6PG) in a reaction that generates NADPH. 11ß-hydroxysteroid dehydrogenase type 1 (HSD11B1) utilizes this NADPH to convert inactive cortisone to cortisol. HSD11B1 inhibitors improve insulin sensitivity whereas G6PC inhibitors are predicted to lower fasting blood glucose (FBG). This study investigated whether G6PC1 and G6PC2 influence G6P flux through H6PD and vice versa. Using a novel transcriptional assay that utilizes separate fusion genes to quantitate glucocorticoid and glucose signaling, we show that overexpression of H6PD and HSD11B1 in the islet-derived 832/13 cell line activated glucocorticoid-stimulated fusion gene expression. Overexpression of HSD11B1 blunted glucose-stimulated fusion gene expression independently of altered G6P flux. While overexpression of G6PC1 and G6PC2 blunted glucose-stimulated fusion gene expression, it had minimal effect on glucocorticoid-stimulated fusion gene expression. In the liver-derived HepG2 cell line, overexpression of H6PD and HSD11B1 activated glucocorticoid-stimulated fusion gene expression but overexpression of G6PC1 and G6PC2 had no effect. In rodents, HSD11B1 converts 11-dehydrocorticosterone (11-DHC) to corticosterone. Studies in wild-type and G6pc2 knockout mice treated with 11-DHC for 5 weeks reveal metabolic changes unaffected by the absence of G6PC2. These data suggest that HSD11B1 activity is not significantly affected by the presence or absence of G6PC1 or G6PC2. As such, G6PC1 and G6PC2 inhibitors are predicted to have beneficial effects by reducing FBG without causing a deleterious increase in glucocorticoid signaling.

Hepatic retinaldehyde deficiency is involved in diabetes deterioration by enhancing PCK1- and G6PC-mediated gluconeogenesis.

Type 2 diabetes (T2D) is often accompanied with an induction of retinaldehyde dehydrogenase 1 (RALDH1 or ALDH1A1) expression and a consequent decrease in hepatic retinaldehyde (Rald) levels. However, the role of hepatic Rald deficiency in T2D progression remains unclear. In this study, we demonstrated that reversing T2D-mediated hepatic Rald deficiency by Rald or citral treatments, or liver-specific Raldh1 silencing substantially lowered fasting glycemia levels, inhibited hepatic glucogenesis, and downregulated phosphoenolpyruvate carboxykinase 1 (PCK1) and glucose-6-phosphatase (G6PC) expression in diabetic db/db mice. Fasting glycemia and Pck1/G6pc mRNA expression levels were strongly negatively correlated with hepatic Rald levels, indicating the involvement of hepatic Rald depletion in T2D deterioration. A similar result that liver-specific Raldh1 silencing improved glucose metabolism was also observed in high-fat diet-fed mice. In primary human hepatocytes and oleic acid-treated HepG2 cells, Rald or Rald + RALDH1 silencing resulted in decreased glucose production and downregulated PCK1/G6PC mRNA and protein expression. Mechanistically, Rald downregulated direct repeat 1-mediated PCK1 and G6PC expression by antagonizing retinoid X receptor α, as confirmed by luciferase reporter assays and molecular docking. These results highlight the link between hepatic Rald deficiency, glucose dyshomeostasis, and the progression of T2D, whilst also suggesting RALDH1 as a potential therapeutic target for T2D.

Theoretical, antioxidant, antidiabetic and in silico molecular docking and pharmacokinetics studies of heteroleptic oxovanadium(IV) complexes of thiosemicarbazone-based ligands and diclofenac.

A series of new heteroleptic oxovanadium(IV) complexes with the general formula [VOL1-6(Dcf)] (1-6), where L1-6 = thiosemicarbazone (TSC)-based ligands and Dcf = diclofenac have been synthesized and characterized. The spectral studies along with the density functional theory calculations evidenced the distorted square-pyramidal geometry around oxovanadium(IV) ion through imine nitrogen and thione sulfur atoms of TSC moiety, and two asymmetric carboxylate oxygen atoms of diclofenac drug. The complexes were evaluated for in vitro antioxidant activity using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,2'-diphenyl-1-picrylhydrazyl (DPPH), hydrogen peroxide (H2O2) and superoxide radical scavenging assays with respect to the standard antioxidant drugs butylated hydroxyanisole (BHA) and rutin. The in vitro antidiabetic activity of the complexes was tested with enzymes such as α-amylase, α-glucosidase and glucose-6-phosphatase. The complexes containing methyl substituent showed higher activity than that containing the nitro substituent due to the electron-donating effect of methyl group. The in silico molecular docking studies of the oxovanadium(IV) complexes with α-amylase and α-glucosidase enzymes showed strong interaction via hydrogen bonding and hydrophobic interactions. The dynamic behavior of the proposed complexes was analyzed by molecular dynamics (MDs) simulations, which revealed the stability of docked structures with α-amylase and α-glucosidase enzymes. The in silico physicochemical and pharmacokinetics parameters, such as Lipinski's 'rule of five', Veber's rule and absorption, distribution, metabolism and excretion (ADME) properties predicted non-toxic, non-carcinogenic and safe oral administration of the synthesized complexes.Communicated by Ramaswamy H. Sarma.

Impact of patient characteristic factors on the dynamics of liver glucose metabolism: Evaluation of multiparametric imaging with dynamic whole-body 18 F-fluorodeoxyglucose-positron emission tomography.

AIMS: To assess the impact of various patient characteristics on the dynamics of liver glucose metabolism using automated multiparametric imaging with whole-body dynamic 18 F-fluorodeoxyglucose (FDG)-positron emission tomography (PET). MATERIALS AND METHODS: We retrospectively enrolled 540 patients who underwent whole-body dynamic FDG-PET. Three quantitative indices representing hepatic glucose metabolism [mean standardized uptake value normalized by lean body mass (SULmean), metabolic glucose rate (kinetic index) and distribution volume (DV)] were measured from multiparametric PET images produced automatically based on the Patlak plot model. Patient characteristics including age, sex, body mass index, fasting time, blood glucose level, and the presence of diabetes mellitus (DM) or hepatic steatosis (HS) were collected. We examined the correlations between the characteristic factors and three quantitative indices using multiple regression analysis. RESULTS: The success rate of kinetic analysis using multiparametric PET imaging was 93.3% (504/540). Hepatic SULmean was significantly correlated with age (p < .001), sex (p < .001) and blood glucose level (p = .002). DV was significantly correlated with age (p = .033), sex (p < .001), body mass index (p = .002), fasting time (p = .043) and the presence of HS (p = .002). The kinetic index was significantly correlated with age (p < .001) and sex (p = .004). In the comparison of the healthy, DM and HS groups, patients with DM had a significantly increased SULmean, whereas patients with HS had a significantly decreased DV. CONCLUSIONS: Our results showed that liver glucose metabolism was influenced by various patient characteristic factors. Multiparametric FDG-PET imaging can be used to analyse the kinetics of liver glucose metabolism in routine clinical practice.

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.

A case of glycogen storage disease type â a with gout as the first manifestation. / 以痛风为首发的糖原贮积症â a型一例.

A 24-year-old male was admitted due to recurrent redness, swelling, fever and pain in the ankle, frequently accompanied by hungry feeling. Dual energy CT scans showed multiple small gouty stones in the posterior edge of the bilateral calcaneus and in the space between the bilateral metatarsophalangeal joints. The laboratory examination results indicated hyperlipidemia, high lactate lipids, and low fasting blood glucose. Histopathology of liver biopsy showed significant glycogen accumulation. The results of gene sequencing revealed the compound heterozygous mutations of the G6PC gene c.248G>A (p.Arg83His) and c.238T>A (p.Phe80Ile) in the proband. The c.248G>A mutation was from mother and the c.238T>A mutation was from father. The diagnosis of glycogen storage disease type Ⅰa was confirmed. After giving a high starch diet and limiting monosaccharide intake, as well as receiving uric acid and blood lipids lowering therapy, the condition of the patient was gradually stabilized. After a one-year follow-up, there were no acute episodes of gout and a significant improvement in hungry feeling in the patient.

Polyphenolic compounds of Phyllanthus amarus Schum & Thonn. (1827) and diabetes-related activity of an aqueous extract as affected by in vitro gastrointestinal digestion.

ETHNOPHARMACOLOGICAL RELEVANCE: Extracts of the aerial part of Phyllanthus amarus have been extensively used in several countries to cure diabetes. No data is available on the impact of gastrointestinal digestion of such crude extracts on their antidiabetic activity. AIM OF THE STUDY: The aim of this study was to identify active fractions and compounds of fresh aerial parts of P. amarus extracted by an infusion method that are responsible for antidiabetic effects occurring at the level of glucose homeostasis. MATERIALS AND METHODS: An aqueous extract was obtained by an infusion method and its polyphenolic composition was analysed by reverse phase UPLC-DAD-MS. The influence of in vitro gastrointestinal digestion was evaluated both on the chemical composition and on the antidiabetic effect of P. amarus infusion extract using glucose-6-phosphatase enzyme inhibition and stimulation of glucose uptake. RESULTS: Analysis of the chemical composition of the crude extract revealed the presence of polysaccharides and various families of polyphenols such as phenolic acids, tannins, flavonoids and lignans. After simulated digestion, the total content of polyphenols decreased by about 95%. Caffeoylglucaric acid derivates and lignans exhibited strong stimulation of glucose uptake similar to metformin with an increase of 35.62 ± 6.14% and 34.74 ± 5.33% respectively. Moreover, corilagin, geraniin, the enriched polysaccharides fraction and the bioaccessible fraction showed strong anti-hyperglycemic activity with about 39-62% of glucose-6-phosphatase inhibition. CONCLUSION: Caffeoylglucaric acid isomers, tannin acalyphidin M1 and lignan demethyleneniranthin were reported for the first time in the species. After in vitro gastroinstestinal digestion, the composition of the extract changed. The dialyzed fraction showed strong glucose-6-phosphatase inhibition.

Treatment of the Neutropenia Associated with GSD1b and G6PC3 Deficiency with SGLT2 Inhibitors.

Glycogen storage disease type Ib (GSD1b) is due to a defect in the glucose-6-phosphate transporter (G6PT) of the endoplasmic reticulum, which is encoded by the SLC37A4 gene. This transporter allows the glucose-6-phosphate that is made in the cytosol to cross the endoplasmic reticulum (ER) membrane and be hydrolyzed by glucose-6-phosphatase (G6PC1), a membrane enzyme whose catalytic site faces the lumen of the ER. Logically, G6PT deficiency causes the same metabolic symptoms (hepatorenal glycogenosis, lactic acidosis, hypoglycemia) as deficiency in G6PC1 (GSD1a). Unlike GSD1a, GSD1b is accompanied by low neutrophil counts and impaired neutrophil function, which is also observed, independently of any metabolic problem, in G6PC3 deficiency. Neutrophil dysfunction is, in both diseases, due to the accumulation of 1,5-anhydroglucitol-6-phosphate (1,5-AG6P), a potent inhibitor of hexokinases, which is slowly formed in the cells from 1,5-anhydroglucitol (1,5-AG), a glucose analog that is normally present in blood. Healthy neutrophils prevent the accumulation of 1,5-AG6P due to its hydrolysis by G6PC3 following transport into the ER by G6PT. An understanding of this mechanism has led to a treatment aimed at lowering the concentration of 1,5-AG in blood by treating patients with inhibitors of SGLT2, which inhibits renal glucose reabsorption. The enhanced urinary excretion of glucose inhibits the 1,5-AG transporter, SGLT5, causing a substantial decrease in the concentration of this polyol in blood, an increase in neutrophil counts and function and a remarkable improvement in neutropenia-associated clinical signs and symptoms.

Genome editing using Staphylococcus aureus Cas9 in a canine model of glycogen storage disease Ia.

Glycogen storage disease type Ia (GSD Ia) is the inherited deficiency of glucose-6-phosphatase (G6Pase), associated with life-threatening hypoglycemia and long-term complications, including hepatocellular carcinoma formation. Gene replacement therapy fails to stably reverse G6Pase deficiency. We attempted genome editing using two adeno-associated virus vectors, one that expressed Staphylococcus aureus Cas9 protein and a second containing a donor transgene encoding G6Pase, in a dog model for GSD Ia. We demonstrated donor transgene integration in the liver of three adult-treated dogs accompanied by stable G6Pase expression and correction of hypoglycemia during fasting. Two puppies with GSD Ia were treated by genome editing that achieved donor transgene integration in the liver. Integration frequency ranged from 0.5% to 1% for all dogs. In adult-treated dogs, anti-SaCas9 antibodies were detected before genome editing, reflecting prior exposure to S. aureus. Nuclease activity was low, as reflected by a low percentage of indel formation at the predicted site of SaCas9 cutting that indicated double-stranded breaks followed by non-homologous end-joining. Thus, genome editing can integrate a therapeutic transgene in the liver of a large animal model, either early or later in life, and further development is warranted to provide a more stable treatment for GSD Ia.

Blood glucose trends in glycogen storage disease type Ia: A cross-sectional study.

BACKGROUND: Glycogen storage disease type Ia (GSDIa) is caused by biallelic pathogenic variants in the glucose-6-phosphatase gene (G6PC) and mainly characterized by hypoglycemia, hepatomegaly, and renal insufficiency. Although its symptoms are reportedly mild in patients carrying the G6PC c.648G>T variant, the predominant variant in Japanese patients, details remain unclear. Therefore, we examined continuous glucose monitoring (CGM) data and daily nutritional intake to clarify their associations in Japanese patients with GSDIa with G6PC c.648G>T. METHODS: This cross-sectional study enrolled 32 patients across 10 hospitals. CGM was performed for 14 days, and nutritional intake was recorded using electronic diaries. Patients were divided according to genotype (homozygous/compound heterozygous) and age. The durations of biochemical hypoglycemia and corresponding nutritional intake were analyzed. Multiple regression analysis was performed to identify factors associated with the duration of biochemical hypoglycemia. RESULTS: Data were analyzed for 30 patients. The mean daily duration of hypoglycemia (<4.0 mmol/L) in the homozygous group increased with age (2-11 years [N = 8]: 79.8 min; 12-18 years [5]: 84.8 min; ≥19 years [10]: 131.5 min). No severe hypoglycemic symptoms were recorded in the patients' diaries. The mean frequency of snack intake was approximately three times greater in patients aged 2-11 years (7.1 times/day) than in those aged 12-18 years (1.9 times/day) or ≥19 years (2.2 times/day). Total cholesterol and lactate were independently associated with the duration of biochemical hypoglycemia. CONCLUSION: Although nutritional therapy prevents severe hypoglycemia in patients with GSDIa with G6PC c.648G>T, patients often experience asymptomatic hypoglycemia.

A caveolin-1 dependent glucose-6-phosphatase trafficking contributes to hepatic glucose production.

OBJECTIVE: Deregulation of hepatic glucose production is a key driver in the pathogenesis of diabetes, but its short-term regulation is incompletely deciphered. According to textbooks, glucose is produced in the endoplasmic reticulum by glucose-6-phosphatase (G6Pase) and then exported in the blood by the glucose transporter GLUT2. However, in the absence of GLUT2, glucose can be produced by a cholesterol-dependent vesicular pathway, which remains to be deciphered. Interestingly, a similar mechanism relying on vesicle trafficking controls short-term G6Pase activity. We thus investigated whether Caveolin-1 (Cav1), a master regulator of cholesterol trafficking, might be the mechanistic link between glucose production by G6Pase in the ER and glucose export through a vesicular pathway. METHODS: Glucose production from fasted mice lacking Cav1, GLUT2 or both proteins was measured in vitro in primary culture of hepatocytes and in vivo by pyruvate tolerance tests. The cellular localization of Cav1 and the catalytic unit of glucose-6-phosphatase (G6PC1) were studied by western blotting from purified membranes, immunofluorescence on primary hepatocytes and fixed liver sections and by in vivo imaging of chimeric constructs overexpressed in cell lines. G6PC1 trafficking to the plasma membrane was inhibited by a broad inhibitor of vesicular pathways or by an anchoring system retaining G6PC1 specifically to the ER membrane. RESULTS: Hepatocyte glucose production is reduced at the step catalyzed by G6Pase in the absence of Cav1. In the absence of both GLUT2 and Cav1, gluconeogenesis is nearly abolished, indicating that these pathways can be considered as the two major pathways of de novo glucose production. Mechanistically, Cav1 colocalizes but does not interact with G6PC1 and controls its localization in the Golgi complex and at the plasma membrane. The localization of G6PC1 at the plasma membrane is correlated to glucose production. Accordingly, retaining G6PC1 in the ER reduces glucose production by hepatic cells. CONCLUSIONS: Our data evidence a pathway of glucose production that relies on Cav1-dependent trafficking of G6PC1 to the plasma membrane. This reveals a new cellular regulation of G6Pase activity that contributes to hepatic glucose production and glucose homeostasis.