The Section on Cellular differentiation conducts research to understand the biology and pathogenesis of GSD-I and G6Pase-beta deficiency and to develop novel therapeutic approaches for these disorders. G6Pase-beta deficiency underlies a congenital neutropenia syndrome in which neutrophils exhibit enhanced ER stress, increased apoptosis, impaired energy homeostasis, and impaired functionality. Granulocyte colony stimulating factor (G-CSF), a cytokine that is widely used to treat neutropenia, can delay neutrophil apoptosis by modulating apoptotic mediators. In G6Pase-beta deficiency G-CSF is used to improve neutrophil counts and decreases the number and severity of bacterial infections but its impact on neutrophil apoptosis and dysfunction is unknown. We hypothesized that in neutrophils of G6Pase-beta-deficient (G6pc3-/-) mice, G-CSF would both delay apoptosis and stimulate glucose uptake, which in turn would improve neutrophil energy homeostasis and functionality. Our results support this hypothesis. We showed that in vitro, G-CSF does modulate apoptotic mediators and delay apoptosis in G6pc3-/- neutrophils, although G6pc3-/- neutrophils still exhibit accelerated apoptosis compared to wild type neutrophils. In contrast, in vivo G-CSF therapy completely corrects neutropenia and normalizes levels of active caspase-3. Moreover, neutrophils from in vivo G-CSF-treated G6pc3-/- mice exhibit increased glucose uptake and elevated intracellular levels of G6P, lactate, and ATP, leading to improved functionality. Together, our results show that G-CSF improves G6pc3-/- neutrophil survival by modulating apoptotic mediators and rectifies function by enhancing energy homeostasis, providing insights into the etiology of neutropenia and neutrophil dysfunction in G6Pase-βdeficiency. We have also delineated the signaling pathways of ER stress and apoptosis in G6pc3-/- neutrophils. We present evidence that the protein kinase-like ER kinase-mediated signaling is one pathway that mediates ER stress and the intrinsic mitochondrial pathway mediates, in part, neutrophil apoptosis in G6pc3-/- neutrophils. In addition to neutropenia and neutrophil dysfunction, G6Pase-beta-deficient patients also exhibits a distinct phenotype of increased visibility of superficial veins, congenital heart defects, and urogenital malformations suggesting that G6Pase-beta deficiency underlies a broader cell dysfunction. The ubiquitous pattern of G6Pase-beta expression suggests that G6Pase-βplays a critical role in non-gluconeogenic tissues where there may be increased demands for glucose. Macrophages play key roles in innate immunity, inflammation, and tissue remodeling. During pregnancy, macrophages also influence the homeostasis of the developing placenta, and are important in preventing premature fetal rejection. Macrophages have increased demand for glucose like neutrophils. Pregnancy is a condition with increased demand for glucose. We hypothesized that the cycling pathway for G6P metabolism (Figure 2) also occurs in macrophages and pregnancy uterus and G6Pase-beta deficiency is associated with impaired energy homeostasis in both macrophages and pregnant uterus, resulting in macrophage dysfunction and pregnancy-associated complications. This was proven to be the case. We showed that murine G6pc3-/- macrophages exhibit impairments in their respiratory burst, chemotaxis, calcium flux, and phagocytic activities. Consistent with a G6P metabolism deficiency, G6pc3-/- macrophages also have a lower glucose uptake, and lower levels of G6P, lactate, and ATP than wild type macrophages. Furthermore, the expression and activation of NADPH oxidase activity is down-regulated. We further showed that during pregnancy, the absence of G6Pase-βactivity also impaired energy homeostasis in the uterus resulting in reduced production of monocyte chemoattractant protein 1 and macrophage colony-stimulating factor, cytokines that recruit macrophages to a site of injury or inflammation. Supporting this, G6pc3-/- macrophages exhibit repressed trafficking in vivo both during an inflammatory response and in pregnancy. The dysfunctional macrophages and impaired uterine energy homeostasis correlate with reduced fertility in G6pc3-/- mothers. GSD-Ia patients deficient in G6Pase-alpha (or G6PC) manifest impaired glucose homeostasis. There is no cure for GSD-Ia, but many of the disease symptoms can be managed or improved using dietary therapies to maintain normoglycemia. While the therapies are sufficiently successful to enable patients to attain near normal growth and pubertal development, the underlying pathological process remains uncorrected. As a result, long-term complications including hepatocellular adenoma (HCA) with malignant potential still persist in GSD-Ia patients. Using our G6pc-/- mouse model of GSD-Ia, we examined the efficacy of liver G6Pase-alpha delivery mediated by AAV8-G6PC-GPE, an AAV serotype 8 vector expressing human G6Pase-alpha directed by the human G6PC promoter/enhancer (GPE) and showed that AAV8-G6PC-GPE-mediated gene transfer completely normalizes hepatic G6Pase-alpha deficiency for at least 24 weeks. A remaining concern with the therapy is the risk of HCA. A recent study showed that HCA develops in 100% of liver-specific G6pc-null mice 78 weeks after gene deletion. Therefore we examined the disease risk for hepatic neoplasia in a long-term study using G6pc-/- mice. We show that AAV8-G6PC-GPE-mediated gene therapy maintains efficacy for at least 70-90 weeks for mice expressing more than 3% of wild type hepatic G6Pase-alpha activity. The treated mice displayed normal hepatic fat storage, normal blood metabolite and glucose tolerance profiles, reduced fasting blood insulin levels, maintained normoglycemia over a 24-hour fast. Ultrasound and histological examinations have shown no hepatic steatosis nor detectable HCA or HCC in the liver of any of these mice. After a 24-hour fast, hepatic G6PT mRNA levels in AAV8-GPE-treated G6pc-/- mice were markedly increased. Since G6PT transport is the rate-limiting step in microsomal G6P metabolism it may explain why the treated G6pc-/- mice could sustain prolonged fasts. The low fasting blood insulin levels and lack of hepatic steatosis may explain the absence of HCA. Our results suggest that G6Pase-alpha gene transfer may offer a therapeutic approach to the management of human GSD-Ia. Blood glucose homeostasis between meals depends upon production of glucose within the ER of the liver and kidney by hydrolysis of G6P into glucose and phosphate (Pi). This reaction depends on coupling the G6PT with G6Pase-alpha. G6PT belongs to the SLC37 family of membrane-bound transporters Only G6PT, also known as SLC37A4, has been characterized, and it acts as a Pi-linked G6P antiporter. The other three SLC37 family members, predicted to be sugar-phosphate:Pi exchangers, have not been characterized functionally. We therefore investigated if any of these proteins might also have a G6P transport activity. Using reconstituted proteoliposomes, we examine the antiporter activity of the other SLC37 members along with their ability to couple with G6Pase-alpha. We show that SLC37A1 and SLC37A2 are ER-associated, Pi-linked antiporters, that can transport G6P. The activity of SLC37A3 is unknown. Unlike G6PT, neither is sensitive to chlorogenic acid, a competitive inhibitor of physiological ER G6P transport, and neither couples to G6Pase-alpha. We conclude that three of the four SLC37 family members are functional sugar-phosphate antiporters. However, only G6PT/SLC37A4 matches the characteristics of the physiological ER G6P transporter, suggesting the other SLC37 proteins have roles independent of blood glucose homeostasis.

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Chou, Janice Y; Jun, Hyun Sik; Mansfield, Brian C (2015) Type I glycogen storage diseases: disorders of the glucose-6-phosphatase/glucose-6-phosphate transporter complexes. J Inherit Metab Dis 38:511-9
Lin, Su Ru; Pan, Chi-Jiunn; Mansfield, Brian C et al. (2015) Functional analysis of mutations in a severe congenital neutropenia syndrome caused by glucose-6-phosphatase-β deficiency. Mol Genet Metab 114:41-5
Kim, Goo-Young; Lee, Young Mok; Cho, Jun-Ho et al. (2015) Mice expressing reduced levels of hepatic glucose-6-phosphatase-α activity do not develop age-related insulin resistance or obesity. Hum Mol Genet 24:5115-25
Lee, Young Mok; Kim, Goo-Young; Pan, Chi-Jiunn et al. (2015) Minimal hepatic glucose-6-phosphatase-α activity required to sustain survival and prevent hepatocellular adenoma formation in murine glycogen storage disease type Ia. Mol Genet Metab Rep 3:28-32
Resaz, Roberta; Vanni, Cristina; Segalerba, Daniela et al. (2014) Development of hepatocellular adenomas and carcinomas in mice with liver-specific G6Pase-* deficiency. Dis Model Mech 7:1083-91
Jun, Hyun Sik; Weinstein, David A; Lee, Young Mok et al. (2014) Molecular mechanisms of neutrophil dysfunction in glycogen storage disease type Ib. Blood 123:2843-53
Lee, Young Mok; Pan, Chi-Jiunn; Koeberl, Dwight D et al. (2013) The upstream enhancer elements of the G6PC promoter are critical for optimal G6PC expression in murine glycogen storage disease type Ia. Mol Genet Metab 110:275-80
Chou, Janice Y; Sik Jun, Hyun; Mansfield, Brian C (2013) The SLC37 family of phosphate-linked sugar phosphate antiporters. Mol Aspects Med 34:601-11
Jun, Hyun Sik; Cheung, Yuk Yin; Lee, Young Mok et al. (2012) Glucose-6-phosphatase-β, implicated in a congenital neutropenia syndrome, is essential for macrophage energy homeostasis and functionality. Blood 119:4047-55
Lee, Young Mok; Jun, Hyun Sik; Pan, Chi-Jiunn et al. (2012) Prevention of hepatocellular adenoma and correction of metabolic abnormalities in murine glycogen storage disease type Ia by gene therapy. Hepatology 56:1719-29

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