Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: SIRT1 maintains bone homeostasis by regulating osteoblast glycolysis through GOT1

doi: 10.1007/s00018-023-05043-9

Figure Lengend Snippet: SIRT1 inhibited GOT1 enzyme activity by catalyzing its lysine deacetylation. A The concentration of Asp in the femurs of the control and cKO mice aged 8 weeks measured by an ELISA Kit (n = 8/group). B The concentration of malate in the femurs of the control and cKO mice aged 8 weeks measured by an ELISA Kit (n = 8/group). C The activity of GOT1 in the femurs of the control and cKO mice aged 8 weeks measured by an ELISA Kit (n = 8/group). D The subcellular localization of SIRT1 and GOT1 in primary osteoblasts. E The protein level and Kac level of GOT1 upon Sirt1 deletion or overexpression in 3T3E1 cells. F The Kac level of GOT1 in Sirt1 knockout 3T3E1 cells upon Sirt1 overexpression. G The Kac level of GOT1 in 3T3E1 cells treated with SRT2104 or EX-527. H The Kac level of GOT1 protein immunoprecipitated from 3T3E1 cells transfected with SIRT1 plasmids or mutant SIRT1-H355A plasmids. I Control and Sirt1 knockout 3T3E1 cells were transfected with Wild-type GOT1 or GOT1-K318R mutant plasmids. The Kac level of GOT1 protein immunoprecipitated from the above cells. J Sirt1-knockout/Got1- knockout 3T3E1 cells were transfected with Wild-type GOT1 or GOT1-K318R mutant plasmids. The GOT1 activity was measured in the above cells. K Sirt1-knockout/Got1- knockout 3T3E1 cells were transfected with Wild-type GOT1 or GOT1-K318R mutant plasmids. The levels of F6P in glycolysis were measured in the above cells. L Sirt1-knockout/Got1- knockout 3T3E1 cells were transfected with Wild-type GOT1 or GOT1-K318R mutant plasmids. The levels of PEP in glycolysis were measured in the above cells. M Sirt1-knockout/Got1- knockout 3T3E1 cells were infected with Wild-type GOT1 or GOT1-K318R mutant virus. The ALP staining of osteoblast mineralization was conducted in the above cells. Data are presented as the mean ± SD, Student’s t test (a, b, c); one-way ANOVA with Tukey’s test (j, k, l), *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: GOT1 activity inhibition To inhibit the activity of GOT1, mice aged 8 weeks were treated with vehicle (saline) and/or 10 mg/kg aminooxyacetic acid (AOA) (MedChemExpress, HY-107994) every other day until dissection at 16 weeks, and osteoblasts were treated with vehicle or 100 μM AOA for 48 h. Statistics Statistical analyses were performed using GraphPad Prism 9.0.

Techniques: Activity Assay, Concentration Assay, Control, Enzyme-linked Immunosorbent Assay, Over Expression, Knock-Out, Immunoprecipitation, Transfection, Mutagenesis, Infection, Virus, Staining

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: SIRT1 maintains bone homeostasis by regulating osteoblast glycolysis through GOT1

doi: 10.1007/s00018-023-05043-9

Figure Lengend Snippet: SIRT1 affected the acetylation level of GOT1 and glycolysis to regulate bone mass. A Schematic of experimental design. The 8-week cKO mice treated with vehicle or AOA until dissection at 16 weeks. B GOT1 activity of 16-week cKO mice treated with vehicle or AOA (n = 8/group). C The concentration of Asp in 16-week cKO mice treated with vehicle or AOA (n = 8/group). D The concentration of malate in 16-week cKO mice treated with vehicle or AOA (n = 8/group). E Representative micro-CT images of the femurs in 16-week cKO mice treated with vehicle or AOA. F Quantification of the BV/TV, Tb.N, Tb.Th, and Tb.Sp from the distal femurs in 16-week cKO mice treated with vehicle or AOA (n = 7/group). G Quantification of the metabolites in glycolysis after AOA treatment in 16-week cKO mice (n = 8/group). H Relative mRNA expression of Runx2, Col1a1, Alpl, and Bglap in Sirt1KO osteoblasts treated with vehicle or AOA (n = 6/group). I Representative images of ALP staining of Sirt1KO osteoblasts treated with vehicle or AOA. J Representative images of Alizarin Red S staining of Sirt1KO osteoblasts treated with vehicle or AOA. K Quantification of caspase-3 activity in Sirt1KO osteoblasts treated with vehicle or AOA (n = 6/group). BV/TV: bone volume fraction; Tb.Th: trabecular thickness; Tb.N: trabecular number; Tb.Sp: trabecular separation; Cort.Th: cortical bone thickness; Tt.Ar: total cortical bone area. Data are presented as the mean ± SD, Student’s t test; *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: GOT1 activity inhibition To inhibit the activity of GOT1, mice aged 8 weeks were treated with vehicle (saline) and/or 10 mg/kg aminooxyacetic acid (AOA) (MedChemExpress, HY-107994) every other day until dissection at 16 weeks, and osteoblasts were treated with vehicle or 100 μM AOA for 48 h. Statistics Statistical analyses were performed using GraphPad Prism 9.0.

Techniques: Dissection, Activity Assay, Concentration Assay, Micro-CT, Expressing, Staining

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: SIRT1 maintains bone homeostasis by regulating osteoblast glycolysis through GOT1

doi: 10.1007/s00018-023-05043-9

Figure Lengend Snippet: Schematic diagram of SIRT1 deacetylating GOT1 to regulate glycolysis in osteoblasts. SIRT1 deletion induced acetylation of GOT1, thereby inhibited the process of glycolysis to decrease bone formation by osteoblasts. The decreased bone formation then inhibited bone resorption. Thus, bone mass decreased

Article Snippet: GOT1 activity inhibition To inhibit the activity of GOT1, mice aged 8 weeks were treated with vehicle (saline) and/or 10 mg/kg aminooxyacetic acid (AOA) (MedChemExpress, HY-107994) every other day until dissection at 16 weeks, and osteoblasts were treated with vehicle or 100 μM AOA for 48 h. Statistics Statistical analyses were performed using GraphPad Prism 9.0.

Techniques:

Journal: Frontiers in Cell and Developmental Biology

Article Title: The β-adrenergic receptor-SGK1 signaling pathway in brown adipocytes protects GOT1 from proteasomal degradation

doi: 10.3389/fcell.2025.1637770

Figure Lengend Snippet: GOT1 protein levels are regulated by the ubiquitin-proteasome pathway (A) A schematic of the malate-aspartate shuttle (MAS) in brown adipocytes. MAS consists of two mitochondrial transporters and two pairs of cytosolic (GOT1 and MDH1) and mitochondrial (GOT2 and MDH2) enzymes. Cold-activated MAS facilitates the transfer of reducing equivalents (especially, electrons from NADH) from the cytosol into the mitochondria. The illustration was created using BioRender ( https://www.biorender.com ). (B) qPCR analysis of Got1 gene expression in BAT during cold exposure. C57BL/6J female mice were housed at 23°C or exposed to 4°C for the times indicated. (C) GOT1 protein expression in cold-exposed BAT. (D) GOT enzymatic activity in BAT homogenates. (E) Degradation of GOT1 protein expressed in HEK293 cells upon treatment with 50 μg/mL of cycloheximide (CHX). A line graph shows the normalized GOT1 protein levels at the indicated time points from three independent experiments. (F) Co-treatment with MG132 attenuates proteasomal degradation of GOT1 in the presence of cycloheximide (CHX). (G) Ubiquitination of GOT1 protein immunoprecipitated from HEK293 cells treated with 10 µM of MG132 for 2 h. (H) Accumulation of GOT1 protein levels in brown adipocytes upon treatment with 10 µM of MG132 for 8 h. Brown adipocytes were stimulated with the β-adrenergic receptor agonist isoproterenol (ISO). All data are presented as the Mean ± SEM. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: Antibodies used were as follows: GOT1 antibody (Pro Sci, #30–379), Myc (9E10) antibody (Cell Signaling, # 2,276), HA antibody (Abcam, #ab9110), NDRG1 (D6C2) antibody (Abcam, #ab 9,408), p-NDRG1 (Thr346) antibody (Abcam, #ab 5,482), RNF34 antibody (Novus, #NBP2-56413), SGK1 antibody (Millipore, # 07–315), SGK2 antibody (Cell Signaling, # 5,595), α-tubulin antibody (Abcam, #ab7291), and β-actin antibody (Sigma, #A5441).

Techniques: Ubiquitin Proteomics, Gene Expression, Expressing, Activity Assay, Immunoprecipitation

Journal: Frontiers in Cell and Developmental Biology

Article Title: The β-adrenergic receptor-SGK1 signaling pathway in brown adipocytes protects GOT1 from proteasomal degradation

doi: 10.3389/fcell.2025.1637770

Figure Lengend Snippet: RNF34 is an E3 ubiquitin ligase for GOT1 (A) A schematic describing the ubiquitin-proteasome-dependent degradation of the protein. E2: E2 ubiquitin-conjugating enzyme, E3: E3 ubiquitin ligase. (B) RNF34-dependent ubiquitination and degradation of GOT1 in HEK293 cells. (C) Rnf34 gene expression in BAT from mice exposed to 4°C for the times indicated. (D) RNF34 protein levels in BAT from mice housed at 23°C or 4°C for 7 days. All data are presented as the Mean ± SEM. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: Antibodies used were as follows: GOT1 antibody (Pro Sci, #30–379), Myc (9E10) antibody (Cell Signaling, # 2,276), HA antibody (Abcam, #ab9110), NDRG1 (D6C2) antibody (Abcam, #ab 9,408), p-NDRG1 (Thr346) antibody (Abcam, #ab 5,482), RNF34 antibody (Novus, #NBP2-56413), SGK1 antibody (Millipore, # 07–315), SGK2 antibody (Cell Signaling, # 5,595), α-tubulin antibody (Abcam, #ab7291), and β-actin antibody (Sigma, #A5441).

Techniques: Ubiquitin Proteomics, Gene Expression

Journal: Frontiers in Cell and Developmental Biology

Article Title: The β-adrenergic receptor-SGK1 signaling pathway in brown adipocytes protects GOT1 from proteasomal degradation

doi: 10.3389/fcell.2025.1637770

Figure Lengend Snippet: The βAR-SGK1 signaling pathway increases GOT1 protein stability in brown adipocytes (A) Effect of kinase inhibitors on GOT1 protein levels. Isoproterenol (ISO)-treated brown adipocytes were co-treated for 2 h with KU0063794, GSK690693, GSK650394, and Go 6,983 to inhibit mTORC2, Akt, SGK, and PKC, respectively. (B) No alteration of Got1 gene expression by the SGK inhibitor GSK650394 (25 µM). (C) Effect of cold stress on the expression of Sgk1 , Sgk2 , and Sgk3 isoforms in BAT. C57BL/6 male mice were housed at 23°C or exposed to 4°C. (D) SGK1 activation stabilizes GOT1. HEK293 cells co-expressing GOT1 and SGK1 S422D or SGK2 S356D were treated with 50 μg/mL of CHX. (E) Sgk1 expression in brown adipocytes treated with vehicle or 10 µM of isoproterenol (ISO) for 4 h. (F) SGK1 S422D -dependent phosphorylation of NDRG1 at Thr346 in HEK293 cells in the absence and presence of MG132. (G) Accumulation of SGK1 protein in brown adipocytes during treatment with 10 µM of MG132. (H) β-adrenergic stimulation of brown adipocytes with isoproterenol induces phosphorylation of NDRG1 at Thr346. (I) Pharmacological inhibition of SGK1 blunts NDRG1 phosphorylation in ISO-stimulated brown adipocytes. (J) SGK1 S422D -dependent phosphorylation of RNF34 in HEK293 cells. (K) SGK1 S422D inhibits RNF34-dependent ubiquitination and degradation of GOT1 in HEK293 cells. (L) SGK1 S422D mitigates the inhibitory effect of RNF34 overexpression on GOT1 in brown adipocytes. (M) A schematic describing the role of SGK1 in enhancing GOT1 protein stability by protecting it from ubiquitin/proteasome-dependent degradation. All data are presented as the Mean ± SEM. ** p < 0.01, # p < 0.0001.

Article Snippet: Antibodies used were as follows: GOT1 antibody (Pro Sci, #30–379), Myc (9E10) antibody (Cell Signaling, # 2,276), HA antibody (Abcam, #ab9110), NDRG1 (D6C2) antibody (Abcam, #ab 9,408), p-NDRG1 (Thr346) antibody (Abcam, #ab 5,482), RNF34 antibody (Novus, #NBP2-56413), SGK1 antibody (Millipore, # 07–315), SGK2 antibody (Cell Signaling, # 5,595), α-tubulin antibody (Abcam, #ab7291), and β-actin antibody (Sigma, #A5441).

Techniques: Gene Expression, Expressing, Activation Assay, Phospho-proteomics, Inhibition, Ubiquitin Proteomics, Over Expression

Journal: Frontiers in Cell and Developmental Biology

Article Title: The β-adrenergic receptor-SGK1 signaling pathway in brown adipocytes protects GOT1 from proteasomal degradation

doi: 10.3389/fcell.2025.1637770

Figure Lengend Snippet: Loss of SGK1 reduces GOT1 protein levels in brown adipocytes without altering its gene expression (A) qPCR analysis of Sgk1 fl/fl and Sgk1 −/− brown adipocytes treated with isoproterenol (ISO) for 4 h. (B) Validation of SGK1 deficiency in Sgk1 −/− brown adipocytes. (C) Loss of SGK1 reduces GOT1 protein levels in ISO-stimulated brown adipocytes. (D) Effects of Sgk1 ablation on mitochondrial respiration in ISO-stimulated brown adipocytes. Left panel: The oxygen consumption rates (OCR) were measured in Sgk1 fl/fl and Sgk1 −/− brown adipocytes after 4 h of ISO stimulation, followed by injections of oligomycin (Oligo), FCCP, and complex III inhibitor antimycin A (AA). Right panel: Total mitochondrial respiration, oligomycin-dependent coupled respiration, and oligomycin-independent leak respiration were determined as described in the Methods section. All data are presented as the Mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Antibodies used were as follows: GOT1 antibody (Pro Sci, #30–379), Myc (9E10) antibody (Cell Signaling, # 2,276), HA antibody (Abcam, #ab9110), NDRG1 (D6C2) antibody (Abcam, #ab 9,408), p-NDRG1 (Thr346) antibody (Abcam, #ab 5,482), RNF34 antibody (Novus, #NBP2-56413), SGK1 antibody (Millipore, # 07–315), SGK2 antibody (Cell Signaling, # 5,595), α-tubulin antibody (Abcam, #ab7291), and β-actin antibody (Sigma, #A5441).

Techniques: Gene Expression, Biomarker Discovery

Journal: Cell Reports

Article Title: The hepatic compensatory response to elevated systemic sulfide promotes diabetes

doi: 10.1016/j.celrep.2021.109958

Figure Lengend Snippet:

Article Snippet: Primary antibodies used were; TST, Rabbit, GeneTex, GTX114858, MPST, Rabbit, Abcam, Ab224043, GOT1, Rabbit, Abcam, ab170950, GSTT1, Rabbit, Proteintech, 15838-1-AP, MAT1A, Rabbit, Abcam, ab129176, BHMT Rabbit, Proteintech, 15965-1-AP, CSAD, Rabbit, Abcam, ab91016, PPCS Rabbit, Atlas Antibodies, HPA031361.

Techniques: Recombinant, Protease Inhibitor, Marker, Staining, Sequencing, Modification, Electron Microscopy, Cholesterol Assay, Multiplex sample analysis, Knock-Out, Gene Expression, Software

Journal: EMBO Reports

Article Title: Glycogenesis and glyconeogenesis from glutamine, lactate and glycerol support human macrophage functions

doi: 10.1038/s44319-024-00278-4

Figure Lengend Snippet: Analysis of the effect of NV-161, CB-839, GOT1i, PEPCKi, MB05032, CHC, GSK2837808, CP-91149, and 6AN on cell viability.

Article Snippet: GOT1i , MedChemExpress , HY-122723.

Techniques: Concentration Assay

Journal: EMBO Reports

Article Title: Glycogenesis and glyconeogenesis from glutamine, lactate and glycerol support human macrophage functions

doi: 10.1038/s44319-024-00278-4

Figure Lengend Snippet: Reagents and tools table

Article Snippet: GOT1i , MedChemExpress , HY-122723.

Techniques: Sequencing, Control, Magnetic Beads, Recombinant, Colorimetric Assay, Staining, Reverse Transcription, Enzyme-linked Immunosorbent Assay, Software, Microscopy, Cytometry, Mass Spectrometry, Imaging, Spectrophotometry