Sirt5 Deficiency Causes Posttranslational Protein Malonylation and Dysregulated Cellular Metabolism in Chondrocytes Under Obesity Conditions

Abstract

Objective: Obesity accelerates the development of osteoarthritis (OA) during aging and is associated with altered chondrocyte cellular metabolism. Protein lysine malonylation (MaK) is a posttranslational modification (PTM) that has been shown to play an important role during aging and obesity. The objective of this study was to investigate the role of sirtuin 5 (Sirt5) in regulating MaK and cellular metabolism in chondrocytes under obesity-related conditions.

Methods: MaK and SIRT5 were immunostained in knee articular cartilage of obese db/db mice and different aged C57BL6 mice with or without destabilization of the medial meniscus surgery to induce OA. Primary chondrocytes were isolated from 7-day-old WT and Sirt5^-/- mice and treated with varying concentrations of glucose and insulin to mimic obesity. Sirt5-dependent effects on MaK and metabolism were evaluated by western blot, Seahorse Respirometry, and gas/chromatography-mass/spectrometry (GC-MS) metabolic profiling.

Results: MaK was significantly increased in cartilage of db/db mice and in chondrocytes treated with high concentrations of glucose and insulin (Glu^hiIns^hi). Sirt5 was increased in an age-dependent manner following joint injury, and Sirt5 deficient primary chondrocytes had increased MaK, decreased glycolysis rate, and reduced basal mitochondrial respiration. GC-MS identified 41 metabolites. Sirt5 deficiency altered 13 distinct metabolites under basal conditions and 18 metabolites under Glu^hiIns^hi treatment. Pathway analysis identified a wide range of Sirt5-dependent altered metabolic pathways that include amino acid metabolism, TCA cycle, and glycolysis.

Conclusion: This study provides the first evidence that Sirt5 broadly regulates chondrocyte metabolism. We observed changes in SIRT5 and MaK levels in cartilage with obesity and joint injury, suggesting that the Sirt5-MaK pathway may contribute to altered chondrocyte metabolism that occurs during OA development.

Keywords: chondrocyte; lysine malonylation; metabolism; osteoarthritis; sirtuin 5.

Figure 1.

Lysine malonylation (MaK) is upregulated in cartilage of db/db mice. (A) Representative immunohistochemical staining for malonyl lysine (MaK) in proximal tibial cartilage of 24-week-old male WT (left) and leptin receptor mutant db/db (right) mice shows a robust upregulation of protein lysine malonylation in db/db chondrocytes. Dashed lines indicate the tidemark, which separates noncalcified cartilage (above) and calcified cartilage (below). (B) IgG negative control using samples from db/db mice. (C) Semiquantitative scoring of malonyl lysine staining intensity in proximal tibial cartilage. Data points show average scoring results for 4 independent biological samples. 0 = no staining; 1 = light staining; 2 = strong staining. Bars are mean ± SD. *P ≤ 0.05.

Figure 2.

High glucose and high insulin treatment (Glu^hiIns^hi) promotes the accumulation of MaK in cultured chondrocytes. (A) Representative image of immunofluorescent staining for MaK (green) and type 2 collagen (COLII, red) in chondrocytes treated with different concentrations of glucose (2.5, 20 mM) with or without insulin (20 nM), yellow arrows indicate cells positive for both COLII and MaK. (B) Quantification of DAPI-normalized MaK staining intensity in chondrocytes treated with different concentrations of glucose (2.5, 10, 20 mM) and insulin (0, 10, 20 nM; n = 9). (C) Table summary of statistical analysis. Data shown as mean ± SD. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

Figure 3.

Effect of aging and joint instability–induced OA on cartilage SIRT5 expression. Representative images of immunohistochemical staining for SIRT5 in articular cartilage from (A) the medial femur and (B) the lateral femur of 22- and 56-week-old mice 8 weeks after DMM surgery. The contralateral knee served as the nonsurgical control (Cont). Scale bar = 50 µm. (C) Semiquantitative scoring of SIRT5 staining intensity in chondrocytes from the femoral and tibial articular cartilage, shown separately for medial and lateral joint compartments. Data points are the average scores of 8 animals per age. 0 = no staining; 1 = light staining; 2 = strong staining.

Figure 4.

Sirt5 deficiency leads to increased MaK in chondrocytes. (A) Representative gel of western blotting analysis of MaK, SIRT5, and Actin in chondrocytes isolated from Sirt5^−/− or WT mice. (B) Quantitative results of densitometry analysis of MaK in chondrocytes normalized to actin. *P = 0.035, primary cells derived from n = 7 animals per genotype.

Figure 5.

Sirt5−/− chondrocytes have lower glycolytic rate. (A) Extracellular acidification rate as a function of time of WT and Sirt5−/− chondrocytes as measured by Seahorse Respirometry analyzer (data normalized to total protein content). Calculated individual parameters: (B) basal glycolysis and (C) compensatory glycolysis. Data shown as mean ± SD. **P < 0.01, n = 5 independent biological replicates per genotype.

Figure 6.

Sirt5^−/− chondrocytes have lower oxygen consumption rate associated with basal respiration and ATP production. (A) Oxygen consumption rate as a function of time of WT and Sirt5^−/− chondrocytes measured by Seahorse Respirometry analyzer (data normalized to total protein content). Calculated individual parameters: (B) basal respiration, (C) ATP production, (D) maximal respiration, and (E) coupling efficiency. Data shown as mean ± SD. **P < 0.01, *P < 0.05, n = 5 independent biological replicates per genotype.

Figure 7.

GC-MS analysis of relative content of metabolites in chondrocytes showed an effect of Sirt5 deficiency and Glu^hiIns^hi condition on chondrocyte cellular metabolism. (A) Principal component analysis (PCA) results based on metabolite abundance values for metabolites detected in both WT and Sirt5^−/− chondrocytes treated with or without Glu^hiIns^hi. (B) Pathway analysis of significantly changed metabolites comparing Sirt5^−/− and WT chondrocytes treated with or without Glu^hiIns^hi (GI), using MetaboAnalyst web-platform (https://www.metaboanalyst.ca) on KEGG database background. (C) Metabolic map (based on KEGG database) of the majority of detected metabolites in WT and Sirt5^−/− chondrocytes. Significantly changed metabolites in the map were marked as *P < 0.05. Icons with solid outlines indicate nontreated groups, icons with dashed outlines indicate GI treated groups.