Abstract Vitamin C (VC, l-ascorbic acid) is an essential nutrient that plays a key role in metabolism and functions as a potent antioxidant in regulating the S-nitrosylation and denitrosylation of target proteins. The precise function of VC deprivation in glucose homeostasis is still unknown. In the absence of L-gulono-1,4-lactone oxidoreductase, an essential enzyme for the last step of VC synthesis, VC deprivation resulted in persistent hypoglycemia and subsequent impairment of cognitive functions in female but not male mouse pups. The cognitive disorders caused by VC deprivation were largely reversed when these female pups were given glucose. VC deprivation-induced S-nitrosylation of glycogen synthase kinase 3β (GSK3β) at Cys14, which activated GSK3β and inactivated glycogen synthase to decrease glycogen synthesis and storage under the feeding condition, while VC deprivation inactivated glycogen phosphorylase to decrease glycogenolysis under the fasting condition, ultimately leading to hypoglycemia and cognitive disorders. Treatment with Nω-Nitro-l-arginine methyl ester (l-NAME), a specific inhibitor of nitric oxide synthase, on the other hand, effectively prevented S-nitrosylation and activation of GSK3β in female pups in response to the VC deprivation and reversed hypoglycemia and cognitive disorders. Overall, this research identifies S-nitrosylation of GSK3β and subsequent GSK3β activation as a previously unknown mechanism controlling glucose homeostasis in female pups in response to VC deprivation, implying that VC supplementation in the prevention of hypoglycemia and cognitive disorders should be considered in the certain groups of people, particularly young females. Keywords: VC, GSK3β, S-nitrosylation, Hypoglycemia, Cognitive disorder Abbreviations: VC, vitamin C; GSK3β, glycogen synthase kinase 3β 1. Introduction Glucose homeostasis in circulation is dynamically regulated by both the glucose production from liver and kidney and glucose usage by peripheral tissues. In the fasted status, glucose is produced by gluconeogenesis and glycogenolysis, whereas glucose is stored by promoting glycogen synthesis and suppressing hepatic glucose output in the postprandial status [[43]1]. Hepatic gluconeogenesis is predominantly determined by glucose-6-phosphatase (G6PC) and phosphoenolpyruvate carboxykinase (PEPCK) encoded by two genes Pck1 (cytosolic form) and Pck2 (mitochondrial form) [[44]2], whereas hepatic glycogenolysis is rate-limited by glycogen phosphorylase (GP). In contrast, glycogen synthesis is an opposite process to glycogenolysis and rate-limited by glycogen synthase (GS), which is inactivated and activated by phosphorylation and dephosphorylation at Ser641, respectively [[45]3]. Hyperglycemia and hypoglycemia are two medical conditions occurring due primarily to the disorder of glycometabolism. Long-lasting hyperglycemia causes diabetes and its complications. Hypoglycemia, an often under-appreciated problem, is usually associated with diabetic glucose-lowering therapy, but other drugs and a variety of conditions also can cause hypoglycemia in people without diabetes [[46]4,[47]5]. Hypoglycemia is related to a variety of symptoms progressing from sweating to seizures and depending on its severity and duration. Untreated hypoglycemia can lead to a series of severe neurological consequences, and impaired cognitive function has potentially deleterious and cumulative long-term effects on intellectual function, especially in young children [[48]6,[49]7]. Protein S-nitrosylation or denitrosylation is a dynamic and reversible post-translational modification by coupling or uncoupling of nitric oxide (NO) with the reactive thiol group of a protein cysteine residue to form or decompose an S-nitrosothiol (SNO), respectively [[50]8,[51]9]. S-nitrosylation or denitrosylation on a cysteine residue of proteins depends on the redox state of cell loci. Vitamin C (VC, l-ascorbic acid), an essential anti-oxidant, not only functions as a scavenger of oxidizing free radicals and prevents the oxidation of other reductants [[52]10,[53]11], but also participates in the denitrosylation of proteins, representing a general mechanism for turning off NO-mediated signaling transduction that is initiated by protein S-nitrosylation [[54]12,[55]13]. Except for its role in protein S-denitrosylation, VC participates in the diabetes-associated glucose metabolism. VC supplement ameliorates symptoms of type 2 diabetes in obese mice with hyperglycemia and diabetic glomerular injury in rats [[56]14], and prevents dexamethasone-induced glucose intolerance as well as tumor necrosis factor-α (TNF-α)-induced insulin resistance [[57]15]. In addition, VC inhibits glucose uptake and lactate production in primary rat adipocytes [[58]16], and especially prevents islet against autoimmunity to lower the risk of type 1 diabetes in children [[59]17]. However, the specific role of VC deprivation in the regulation of glycometabolism remains unknown. Though persistent deprivation of VC leads to scurvy in a certain condition, there are certain groups of people encountering the risk of VC deficiency, including people who are addicted to drugs or alcohol, people who live on a low income, people with medical conditions such as Crohn's disease or ulcerative colitis, pediatric patients with advanced chronic kidney disease, children with severely restricted diets attributable to psychiatric or developmental problems, older people who eat a less varied diet and smokers [[60][18], [61][19], [62][20], [63][21], [64][22]]. For instance, a 17-year-old male who suffers from hereditary fructose intolerance exhibits severe VC deficiency (serum VC < 10 μM; normal range: 26–84 μM), hepatomegaly, proximal tubular dysfunction, and hypoglycemia as well [[65]23]. In the present study, we attempted to assess the role of VC deprivation in glucose metabolism in mice absent of L-gulono-1,4-lactone oxidoreductase (Gulo^−/−), which catalyzes the last step of VC biosynthesis. We found that VC deprivation induced the S-nitrosylation of glycogen synthase kinase 3β (GSK3β) at Cys14, which activated GSK3β and in turn inactivated GS to decrease the glycogen synthesis and storage under fed conditions, and meanwhile VC deprivation inactivated GP to decrease the glycogenolysis under fasting conditions, eventually leading to the hypoglycemia and consequent cognitive disorders. 2. Research design and methods 2.1. Animal care and handling Gulo^−/− mice on the C57BL/6 genetic background was generated by transcription activator-like effector nuclease (TALEN) technique as described previously [[66]24]. Gulo^−/− mice could not synthesize VC by themselves (serum VC: ∼10 μM) and were maintained with tap water containing 3.3 g/L of VC (serum VC: ∼60 μM) [[67]14]. All mice were housed in a specific pathogen free animal facility of Zhejiang University and allowed free access to regular rodent chow or VC-free rodent chow (Trophic Animal Feed High-tech Corporation, Nantong, China). All animal cares and handling procedures (Protocol No. 20180226-003) were approved by the Institutional Animal Care and Use Committee of Zhejiang University. 2.2. Experimental designs Female and male Gulo^−/− mice were maintained with tap water containing 3.3 g/L of VC, and crossed to generate Gulo^−/− pups. Pups were all weaned at day 21 after birth and 22-day-old Gulo^−/− pups were then randomly and evenly divided into four groups: (1) male or female pups supplemented with 3.3 g/L of VC (VC+), (2) male or female pups deprived of VC (VC-). Alternatively, 22-day-old female Gulo^−/− pups supplemented with or without 3.3 g/L of VC, were peritoneally injected with or without l-NAME at 0.8 mg/10 body weight, once daily, for five consecutive weeks. All pups were allowed free access to VC-free rodent chow. After fasted for 16 h, blood glucose was measured by a glucometer (Onetouch Ultra, Johnson, LifeScan Europe, UK). Meanwhile, blood samples were harvested and centrifuged at 3000 g for 20 min. Serum was stored at −80 °C until analysis. In addition, after livers were removed and weighed, the left lateral lobes were fixed with 4% formaldehyde and the remaining lobes were all frozen in liquid nitrogen and then stored at −80 °C. 2.3. Pyruvate tolerance test and intraperitoneal glucose tolerance test Pyruvate tolerance test (PTT) and intraperitoneal glucose tolerance test (IPGTT) were performed in female pups after VC deprivation for 3 (6-week-old) and 5 weeks (8-week-old), respectively. Pups were fasted for 16 h, and then intraperitoneally (i.p.) injected with sodium pyruvate (Sigma) at 1.5 g/kg body weight for PTT or with d-glucose (Sigma) at 1 g/kg body weight for IPGTT, as previously described [[68]25]. Blood glucose was measured at 0, 15, 30, 60, 90, and 120 min after the injection. 2.4. Biochemical and histological analyses Blood samples were used to determine the levels of insulin or glucagon using a mouse insulin or glucagon enzyme-linked immunosorbent assays (ELISA) kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's instructions, respectively. The Periodic Acid-Schiff (PAS) and immunohistochemistry staining were performed as previously described [[69]14,[70]26]. The primary antibodies against p-Ser641-GS (1:200 dilution, ET1602-13; HuaAn Biotechnology, Hangzhou, China), p-Ser15-glycogen phosphorylase L (PYGL; 1:200 dilution, ab227043; Abcam, Cambridge, UK), and p-Ser9-GSK3β (1:50 dilution, ab131097; Abcam) were used in the experiments. 2.5. Isolation and culture of mouse primary hepatocytes Primary mouse hepatocytes were isolated from female Gulo^−/− pups with deprivation of VC for 3-week by digesting the livers with type II collagenase (CAT [71]LS004176; Worthington, Lakewood, CO) as described previously [[72]27]. Isolated hepatocytes were seeded into 6-well-plate covered with type I collagen (CAT 354236; Corning, Glendale, AZ) and successfully attached at 2 h post-inoculation. VC was added to stimulate primary mouse hepatocytes for the indicated times, and then cells were harvested for western blotting analyses. 2.6. Cell cultures and transfection Human hepatocytes L02 cells were gifted from Prof. Zhi Chen at the First Affiliated Hospital, Zhejiang University School of Medicine, and maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (Life Technologies, Carlsbad, CA) and 1% penicillin-streptomycin (Sigma). Human hepatocarcinoma HepG2 cells from Chinese Academy of Sciences (Shanghai, China) were cultured in Modified Eagle Medium (MEM) containing 10% FBS, 1% l-glutamine (Invitrogen, Grand Island, NY), 1% non-essential Amino Acids (Invitrogen), and 1% sodium pyruvate (Invitrogen). VC (Sigma), N6022 (S7589; Selleckchem, Huston, TX), and Nω-Nitro-l-arginine methyl ester (l-NAME, S2877; Selleckchem) were used to stimulate L02 and HepG2 cells. Cell lines were all incubated at 37 °C with 5% CO[2]. pCMV-3 × Flag-GSK3β construct was obtained from MiaoLingBio (Wuhan, China) and verified by DNA sequencing. Cys14Ser (C14S) mutation of GSK3β was introduced by using a KOD-Plus-Mutagenesis Kit (SMK-101; Toyobo, Osaka, Japan) according to the manufacturer's instruction. Transient transfection was performed by using Hieff Trans^TM Liposomal Transfection Reagent (40802ES03; Yeasen Biotechnology, Shanghai, China) as previously described [[73]28]. 2.7. In vitro S-Nitrosylation assay In vitro S-nitrosylation assays were performed using a Pierce^TM S-nitrosylation Western Blot Kit (90105; Thermo scientific, Waltham, MA) with modification. All reactions were performed in the dark place to avoid light exposure after immunoprecipitation and enrichment of GSK3β. Protein lysates at 1 mg/mL were prepared in 100 μl HENS buffer (Sigma) and S-nitrosylated cysteines were labelled with a non-biological iodoTMT^TM Reagent as described previously [[74]29]. 2.8. Western blotting Proteins from livers, primary hepatocytes, and cell lines were extracted by cell lysis buffer (Beyotime Biotechnology, Shanghai, China) containing protease inhibitor and phosphatase inhibitors (Bimake, Houston, TX). 50 μg protein was separated by 8–12% SDS-PAGE, transferred onto a nitrocellulose membrane, and incubated with primary antibodies at 4 °C overnight. The following primary antibodies were used in the experiments: anti-Pck1 (1:1000 dilution, ab70358), anti-p-Forkhead box O1a (Foxo1a; 1:1000 dilution, ab131339), anti-p-PYGL (1:1000 dilution, ab227043), and anti-p-GSK3β (1:1000 dilution, ab131097) were purchased from Abcam, anti-PYGL (1:1000 dilution, NBP1-86182) was purchased from Novus Biologicals (Centennial, CO), anti-Flag (1:2000 dilution, TA180144) was purchased from Origene (Rockville, MD), anti-Pck2 (1:1000 dilution, ET7107-29), anti-Foxo1a (1:1000 dilution, ET1608-25), anti-p-GS (1:1000 dilution, ET1602-13), anti-GS2 (1:1000 dilution, ER1909-76), anti-GSK3β (1:1000 dilution, ET1607-71), anti-Gapdh (1:5000 dilution, R1210-1) and anti-α-tubulin (1:2000 dilution, [75]ER130905) were purchased from HuaAn Biotechnology. IRDye 680 (926–68070; LI-COR, Lincoln, NE) or 800s antibody (926–32211, LI-COR) was used as the second antibodies. Immunoreactive bands were visualized by Odyssey Infrared Imaging System (LI-COR) and semi-quantified by ImageJ (NIH, Bethesda, MD). The phosphorylated protein was normalized to its total protein, whereas total protein was normalized to either Gapdh or α-tubulin. The first band was defined as 1. 2.9. Morris water maze Female Gulo^−/− pups at 3 weeks old randomly received tap water containing 3.3 g/L of VC, 2 g/L of glucose, or neither VC nor glucose for three consecutive weeks. Alternatively, female Gulo^−/− pups supplemented with or without 3.3 g/L of VC, were peritoneally injected with or without l-NAME at 0.8 mg/10 body weight, once daily, for four consecutive weeks. These groups of pups were then subjected to the tests for learning and memory abilities by using a Morris water maze as described previously [[76]30]. 2.10. Statistical analysis Numeral data from animal and cell experiments were expressed as Mean ± SEM or Mean ± SD. Statistical analyses were conducted by one-way ANOVA and Dunnett's multiple comparison tests or by Welch's t-test (Graphpad Software Inc., La Jolla, CA). Statistical significance was assessed at p < 0.05 and p < 0.01. All experiments were repeated independently three times with similar results, and the representative data were shown. 3. Results 3.1. VC deprivation lowered the fasting serum glucose in a sex-specific manner In Gulo^−/− mice, VC deprivation for 2 weeks reduced serum VC by 85%, while a 3.3 g/L VC supplement in drinking water maintained the normal serum VC level (approximately 60 μM) [[77]14,[78]15]. To determine the role of VC deprivation in regulating fasting serum glucose (FSG), female and male Gulo^−/− pups were started on VC deprivation at 3 weeks old, and FSG levels were measured once a week for 5 weeks. We chose 5 weeks as the observational period because, after 5 weeks of VC deprivation, some Gulo^−/− pups died. FSG levels in female Gulo^−/− pups dropped significantly at week 3 after VC deprivation and remained low until week 5 after VC deprivation ([79]Fig. 1A). FSG levels in male Gulo^−/− pups, on the other hand, dropped significantly at week 5 after VC deprivation ([80]Fig. 1B). When VC-deprived female Gulo^−/− pups were compared to VC-supplemented female Gulo^−/− pups, the area under the curve (AUC) of FSG values from weeks 3–5 was significantly reduced by 24%. ([81]Fig. 1C). Although the AUC of FSG values decreased by 11% from week 3–5 in VC-deprived male Gulo^−/− pups compared to VC-supplemented male Gulo^−/− pups, there was no statistical difference in this decrease ([82]Fig. 1D). Thus, VC deprivation reduced FSG in female Gulo^−/− pups but not in male Gulo^−/− pups. Fig. 1. [83]Fig. 1 [84]Open in a new tab Female Gulo^−/− pups had lower fasting serum glucose levels after VC deprivation. Male or female pups at 22 days old were subjected to VC supplementation (VC+) or deprivation (VC-) for 5 weeks. Before blood isolation and serum glucose measurement, each group of pups was starved for 16 h. (A and B) Fasting serum glucose (FSG) levels in male and female pups after VC deprivation for the indicated times. (C and D) FSG values for the area under the curve (AUC) were calculated during a 3 to 5-week period of VC deprivation. (E and F) The average food intake of 24 h per pup relative to body weight was calculated during the 3 weeks of VC deprivation. Welch's t-test, n = 6, mean SD; *p < 0.05, **p < 0.01 versus VC+. We measured the average food intake of 24 h relative to body weight in female and male Gulo^−/− pups after 3 weeks of VC deprivation to rule out the possibility that insufficient food intake caused hypoglycemia. Female and male Gulo^−/− pups with VC deficiency had significantly lower average food intakes in equal measure over 24 h ([85]Fig. 1E and F). Similarly, from weeks 3–5 after VC deprivation, the body weights of VC-deprived female and male Gulo^−/− pups were significantly reduced to the same extent ([86]Figs. S1A and B). Also, when both female and male Gulo^−/− pups were denied VC for 3 weeks, their average 24-h water intake was reduced indiscriminately concerning body weight. ([87]Figs. S1C and D). We compared FSG levels and body weights in wild-type pups and Gulo^−/− pups supplemented with 3.3 g/L VC to rule out the possibility that hypoglycemia in female Gulo^−/− pups was caused by genetic manipulation or knockout of the Gulo gene. Compared to female wild-type pups, genetic manipulation or knockout of the Gulo gene did not affect FSG levels or body weights in female Gulo^−/− pups supplemented with 3.3 g/L VC ([88]Figs. S2A and B). Also, measurements of fasting serum insulin and glucagon levels in female Gulo^−/− pups consistently showed that VC deprivation for 4 weeks did not affect serum insulin or glucagon levels ([89]Figs. S3A and B). Finally, measurements of the liver coefficient revealed that 5 weeks of VC deprivation significantly reduced the liver coefficient in both female and male Gulo^−/− pups, though the decrease was more pronounced in males than females ([90]Figs. S4A and B). In Gulo^−/− pups, VC deprivation reduced FSG in a sex-dependent manner, but not in a food intake-, genetic manipulation-, or hormone-dependent manner. 3.2. VC deprivation affected the glycometabolism in female Gulo^−/− pups We used Tandem Mass Tag (TMT)-based quantitative proteomics analyses in the livers of female Gulo^−/− pups after 3 weeks of VC deprivation to investigate the potential mechanism underlying VC deprivation-induced hypoglycemia. There were 5651 proteins found with a normal distribution ([91]Supplementary Table 1). Differentially expressed proteins (DEPs) were defined as proteins with a fold change of >1.2 or <0.833 (p < 0.05). 406 DEPs were found among the 5651 proteins, with 156 and 250 of them significantly up-regulated and down-regulated, respectively ([92]Supplementary Table 2). The volcano plot displayed the statistical results of protein quantification. ([93]Fig. 2A). Similarly, pathway enrichment analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) was used to determine the functions of DEPs. DEPs were found in 276 KEGG pathways in VC-deficient pups ([94]Supplementary Table 3). We focused on the metabolic and glucagon signaling pathways among the top 20 significant pathways ([95]Fig. 2B) to investigate the mechanism underlying VC deprivation-induced hypoglycemia. Hierarchical clustering analysis of DEPs indicated that the glucagon signaling pathway was composed of 7 DEPs ([96]Fig. 2C), including Acetyl-CoA carboxylase 1 (Acaca), Acetyl-CoA carboxylase 2 (Acacb), Phosphoglycerate mutase 2 (Pgam2), Solute carrier family 2 facilitated glucose transporter member 2 (Slc2a2), GP of muscle form (fold change = 0.4338), GP of brain form (fold change = 0.8146), and GP of the liver form (fold change = 0.8328). In female Gulo^−/− pups, VC deprivation affected the glucose metabolism signaling pathway. Fig. 2. [97]Fig. 2 [98]Open in a new tab In female Gulo^−/− pups, GP was significantly down-regulated after VC deprivation. TMT-based comparative proteomics analyses for female pups' livers supplemented or depleted of VC for 3 weeks. (A) A volcano plot of DEPs in liver tissues. All proteins were plotted on the x-axis with log2 fold change and log10 (the sum of intensity in two samples) on the y-axis. The red dots in the upper right (ratio>1.2) and blue dots in the upper left (ratio<0.833) sections with p < 0.05 represent proteins that were significantly up-or down-regulated between groups. Gray dots represented proteins that were identical in the two compared groups. (B) KEGG pathway enrichment analysis of the top twenty enrichment scores for DEPs. The pathways were depicted on the y-axis. The x-axis displayed the number of DEPs in each pathway. (C) Hierarchical clustering of DEPs in the glucagon signaling pathway. Each column represented a tissue sample, and each line represented a protein that was differentially expressed. The expression levels of DEPs were indicated by a color scale ranging from blue (low) to red (high). The colors red and blue represent up-and down-regulation, respectively. The case represents female pups with VC deficiency, while the control represents female pups with VC supplementation. (For interpretation of the references to color in this figure legend, the reader is referred