Fig 2: SIRT1 and SIRT6 silencing triggers inflammation and enhances glycolysis in hASCs, whereas enhancing glycogen deposition reduces SIRT1 and SIRT6 protein expression. (a,b) Relative gene expression of SIRT1 (a) and SIRT6 (b) and inflammatory markers (IL1B, IL6, CCL2, and TNFA) in lean adult (LA) hASCs silenced with siSIRT1 (a) (n = 6) or siSIRT6 (b) (n = 5) versus control hASCs. c,d) Relative gene expression of SIRT1 (c) or SIRT6 (d) and glycolytic markers (HK2, PFKM, and PDK4), tricarboxylic acid cycle enzymes (LDHB, SDHB, and OGDH), glycogen synthesis and degradation enzymes (GYS, PYGL, and GBE1) and glucose transporters (SLC2A1 and SLC2A3) in siSIRT1 (c) (n = 6) or siSIRT6 (d) (n = 5) hASCs versus control hASCs. (e–g) protein targeting to glycogen (PTG) was overexpressed in hASCs using an adenoviral transduction system. (e,f) LA hASCs overexpressing PTG versus those overexpressing GFP (control) were analyzed for glycogen deposition (e) (n = 8) and p-GS, p-PYGL, SIRT1, SIRT6, p-AMPK, PTG, and GFP protein expression (f) (n = 3–16). (g) Gene expression of PPP1R3C, SIRT1, SIRT6 and inflammatory markers (IL1B, IL6, CCL2, and TNFA) was determined in hASCs overexpressing PTG versus those overexpressing GFP (control) (n = 5–8). (h–j) Proliferation determined by MTT assay (h) (n = 5) and adipocyte differentiation analyzed by Oil Red O staining (i) (n = 3) and gene expression of adipocyte differentiation markers (j) (n = 3) were assessed in hASCs upon PTG overexpression versus those overexpressing GFP (control). (k–m) Representative immunoblots and densitometric analysis of SIRT1, p-GS, p-PYGL and p-AMPK (k) (n = 3–6), glycogen content (l) (n = 6) and proliferation (m) (n = 5) in hASCs transfected with siSIRT1 versus control hASCs. (n–p) Representative immunoblots and densitometric analysis of SIRT6, p-GS, p-PYGL and p-AMPK (n) (n = 3–5), glycogen content (o) (n = 4) and proliferation (p) (n = 5) in hASCs transfected with siSIRT6 versus control hASCs. For immunoblots, GAPDH was used as the loading control. Densitometric analyses are presented in arbitray units (a.u.). Data are shown as mean ± SEM; Two-tailed paired Student's t test, p < 0.05 (*)

Fig 3: Female Gulo-/- pups' glycogen synthesis and glycogenolysis were affected by VC deprivation. Female Gulo-/- pups (six pups per group) were supplemented with 3.3 g/L VC or denied VC in their drinking water for 3, 4, or 5 weeks. Three livers were chosen at random for the following experiments. (A, B, E, F, I, J) Western analyses and semi-quantification of p-Ser641-GS, GS2, p-Ser15-PYGL, and PYGL. (C, D, G, H, K, L) Immunohistochemistry staining and semi-quantification for p-Ser641-GS and p-Ser15-PYGL. (C and D) Negative controls were used to assess the specificity of the immunohistochemistry staining and to rule out false-positive staining reactions. Welch's t-test, n = 3, mean SD; *p < 0.05, **p < 0.01 versus VC+. Square frames define the magnified regions.

Fig 4: Pharmacological blockade of glycogen utilization (PYGL) impairs chondrosarcoma tumor growth in vivo, reduces proliferation, and induces cellular senescence in glycogen-deprived tumors15-day treatment of PDX tumors with 50 mg/kg CP-91149 resulted in a reduction in (A) tumor size (n = 4), (B) tumor growth (n = 4), (C) tumor weight (n = 14), and (D) tumor volume (n = 14).(E) BrdU staining (n = 3) of CP-91149-treated tumors revealed a reduction in BrdU incorporation, suggesting a reduction in proliferation, and an increase in DEC1 (n = 3) and p21 (n = 3) staining, suggesting glycogenolysis blockade induces cellular senescence. p value = Student’s t test p < 0.05, an asterisk (*) indicates that significant p values are shown. Means and error bars representing standard deviations are shown. Scale bars: 50 µm in black.

Fig 5: Glycogen levels are elevated in mutant IDH chondrosarcoma patient tissue and in the mutant Idh1-KI R132Q fetal growth plate(A) Transmission electron microscopy (TEM) images of IDH non-mutant chondrosarcoma cells (n = 4) in vitro display absent glycogen granules. Organelles distinct from glycogen granules shown in magnified insets labeled as follows: M, mitochondria; L, lysosome; ER, rough endoplasmic reticulum. TEM images: 7× magnification, inset images: 70× magnification.(B) TEM images of mutant IDH chondrosarcoma cells (n = 7) display glycogen pools, asterisks denote aggregates of glycogen pools in mutant IDH cells, and arrows in magnified insets indicate glycogen pools. Glycogen appears as closely packed circular granules in mutant IDH chondrosarcoma patient cells. Images: 7× magnification, inset images: 70× magnification.(C) Glycogen quantification from pulverized patient-derived xenograft chondrosarcoma tissues display an elevation of glycogen in mutant IDH1 (n = 17) and IDH2 (n = 8) tumors compared with non-mutant tumors (n = 8). One-way ANOVA confirms significant statistical difference of glycogen levels between tumor genotypes (F(2,30) = 6.150, p = 0.0058). Tukey’s multiple comparisons test indicates that the mean values of glycogen in mutant IDH1 (p = 0.0107) and mutant IDH2 groups (p = 0.0109) were significantly higher than in non-mutant tumors.(D) PAS staining identified glycogen deposits in mutant IDH1 (n = 4) and IDH2 (n = 4) patient chondrosarcomas compared with non-mutant tumors (n = 5). PAS-D staining displays dissolution of glycogen deposits in mutant IDH tumors, thus confirming the presence of glycogen deposits. Arrows in magnified insets indicate glycogen deposits in cytoplasm of cells. Images: 40× magnification, inset images: 60× magnification.(E) Quantification of PAS-stained area (µm2), normalized to total number of cells, and of PAS-D-stained area shows an elevation of glycogen deposits in mutant IDH1 (n = 4) and IDH2 (n = 4) chondrosarcomas compared with non-mutant tumors (n = 5). One-way ANOVA confirms significant statistical difference of glycogen levels between tumor genotypes (F(2,10) = 7.537, p = 0.0101). Tukey’s multiple comparisons test indicates mean values of glycogen in mutant IDH1 (p = 0.0380) and mutant IDH2 groups (p = 0.0123) were significantly higher than non-mutant tumors.(F) Glycogen deposits in Col2a1Cre; Idh1LSL/wt E18.5 growth plates (n = 9) shown by PAS staining. Arrows in magnified insets indicate glycogen in cell cytoplasm. Glycogen deposits minimally in Col2a1Cre; Idh1wt/wt control growth plates (n = 8).(G) Quantification of PAS-stained area (µm2), normalized to total number of cells, and PAS-D-stained area (n = 8).(H) GYS1 staining is elevated in Col2a1Cre; Idh1LSL/wt growth plates compared with Col2a1Cre; Idh1wt/wt growth plates (n = 5).(I) Quantification of GYS1 staining from hypertrophic to resting zones (n = 5).(J) PYGL staining is unchanged in Col2a1Cre; Idh1LSL/wt and Col2a1Cre; Idh1wt/wt growth plates (n = 6).(K) Quantification of PYGL staining from hypertrophic to resting zones (n = 6).(L) Gene expression levels of glycogen genes are elevated upon Idh1 mutation induced by adenovirus Cre recombinase transfection (n = 3). Relative gene expression compared with adenovirus GFP control group was calculated and normalized to ß-actin using the 2-??Ct method. One-way ANOVA with Tukey’s multiple comparisons test = p < 0.05. p value = Student’s t test p < 0.05, an asterisk (*) indicates that significant p values are shown. Means and error bars representing standard deviations are shown. Scale bars: 2 µm in white, 100 µm in black, and 10 µm in blue. Magnification: whole growth plate images: 13× magnification, inset images: 60× magnification.