Sodium butyrate activates HMGCS2 to promote ketone body production through SIRT5-mediated desuccinylation

doi:10.1007/s11684-022-0943-0

Front. Med.

2023, Vol. 17

Issue (2) : 339-351 https://doi.org/10.1007/s11684-022-0943-0

RESEARCH ARTICLE

Sodium butyrate activates HMGCS2 to promote ketone body production through SIRT5-mediated desuccinylation

Yanhong Xu¹, Xiaotong Ye², Yang Zhou², Xinyu Cao³, Shiqiao Peng³, Yue Peng⁴, Xiaoying Zhang⁵, Yili Sun⁶, Haowen Jiang⁶, Wenying Huang⁴, Hongkai Lian⁵, Jiajun Yang¹, Jia Li⁶, Jianping Ye^5,⁷(

)

¹. Neurology Department, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai 201306, China
². National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai 201306, China
³. Shanghai Diabetes Institute, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai 200233, China
⁴. School of Physical Education, Jiangxi Normal University, Nanchang 330022, China
⁵. Metabolic Disease Research Center, Zhengzhou University Affiliated Zhengzhou Central Hospital, Zhengzhou 450007, China
⁶. State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
⁷. Center for Advanced Medicine, College of Medicine, Zhengzhou University, Zhengzhou 450007, China

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Abstract

Ketone bodies have beneficial metabolic activities, and the induction of plasma ketone bodies is a health promotion strategy. Dietary supplementation of sodium butyrate (SB) is an effective approach in the induction of plasma ketone bodies. However, the cellular and molecular mechanisms are unknown. In this study, SB was found to enhance the catalytic activity of 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), a rate-limiting enzyme in ketogenesis, to promote ketone body production in hepatocytes. SB administrated by gavage or intraperitoneal injection significantly induced blood β-hydroxybutyrate (BHB) in mice. BHB production was induced in the primary hepatocytes by SB. Protein succinylation was altered by SB in the liver tissues with down-regulation in 58 proteins and up-regulation in 26 proteins in the proteomics analysis. However, the alteration was mostly observed in mitochondrial proteins with 41% down- and 65% up-regulation, respectively. Succinylation status of HMGCS2 protein was altered by a reduction at two sites (K221 and K358) without a change in the protein level. The SB effect was significantly reduced by a SIRT5 inhibitor and in Sirt5-KO mice. The data suggests that SB activated HMGCS2 through SIRT5-mediated desuccinylation for ketone body production by the liver. The effect was not associated with an elevation in NAD⁺/NADH ratio according to our metabolomics analysis. The data provide a novel molecular mechanism for SB activity in the induction of ketone body production.

Keywords sodium butyrate succinylation HMGCS2 ketogenesis SIRT5

Corresponding Author(s): Jianping Ye

Just Accepted Date: 24 October 2022 Online First Date: 06 January 2023 Issue Date: 26 May 2023

Cite this article:

Yanhong Xu,Xiaotong Ye,Yang Zhou, et al. Sodium butyrate activates HMGCS2 to promote ketone body production through SIRT5-mediated desuccinylation[J]. Front. Med., 2023, 17(2): 339-351.

URL:

https://academic.hep.com.cn/fmd/EN/10.1007/s11684-022-0943-0
https://academic.hep.com.cn/fmd/EN/Y2023/V17/I2/339

Fig.1 General changes in protein succinylation status in mouse liver tissue. (A,B) General results of succinylation proteomics of liver tissue at 0.5 h after SB intraperitoneal injection (2.5 g/kg). (C,D) Succinylation profile of liver proteins after SB injection at 1.2-fold as threshold. NS, normal saline. (E,F) Subcellular locations of proteins with up- or downregulated succinylation. (G) Clusters of Orthologous Groups (COG) analysis of succinylation differential proteins. The livers of 3 mice were pooled to form one sample, and 3 samples were analyzed in each group in the proteomics study.

Tab.1 List of mitochondrial proteins at twofold threshold in succinylation change

Fig.2 Succinylation reduction in the key enzyme of ketogenesis pathway by SB treatment. (A) KEGG pathway from proteins with downregulated succinylation in the liver of SB-treated mice. (B) Succinylation status at different lysine residues in 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2). (C) Acetylation of HMGCS2 protein at lysin residues K243 and K310. * P < 0.05.

Fig.3 SB induces ketogenesis. (A) Blood BHB levels in the gavage model (SB at 2.5 g/kg, n = 6). (B) BHB concentration in urine collected for 4 h after gavage (n = 6). (C) Total amount of BHB in the urine (n = 6). (D) Blood BHB levels in the intraperitoneal injection model (SB 2.5 g/kg, control n = 10; SB n = 8). BHB concentration was detected with a ketone meter. (E) Liver BHB levels determined by GC/MS method at 1 h of SB injection (2.5 g/kg, control n = 9; SB n = 7). (F) Liver BHB detected with the fluorometric assay kit. The assay was done in liver tissue at 1 h of SB (2.5 g/kg) injection (n = 6). * P < 0.05, ** P < 0.01, ***P < 0.001.

Fig.4 SB has no effect on the protein level of HMGCS2. (A,B) HMGCS2 protein in the liver tissue determined by Western blot (n = 3). (C,D) The protein level of HMGCS2 in the liver at 5 time points of SB treatment in Western blot (n = 4). (E,F) Protein succinylation and SIRT5 protein in the liver tissue at 30 min after SB injection in Western blot. SB was administrated at 2.5 g/kg by intraperitoneal injection. ** P < 0.01, ***P < 0.001.

Fig.5 SB promotes ketogenesis in primary hepatocytes and reduces the level of succinylation. (A) BHB production by the primary hepatocytes. BHB in the cell supernatant was detected with the BHB meter at different dosages and time points of SB treatment (n = 3). (B,C) Protein succinylation profile in the primary hepatocytes after SB treatment. (D,E) The protein levels of SIRT5 and HMGCS2 determined by Western blot. (F) Histone acetylation at H3K27 in the liver tissue of SB-treated mice as an indicator of HDAC inhibition. * P < 0.05, ** P < 0.01, *** P < 0.001.

Fig.6 SB increases the interaction between HMGCS2 and SIRT5. IP was conducted in the homogenate of liver tissues that were obtained at 0.5 h of SB (2.5 g/kg) intraperitoneal injection. (A,B) HMGCS2 and SIRT5 interaction was investigated in the IP products of HMGCS2 (n = 4). (C,D) HMGCS2 and SIRT5 interaction was studied in the IP products of SIRT5 (n = 3). (E) Levels of NAD⁺ and NADH and their ratio in the liver tissue at 0.5 h of SB injection. NAD⁺ and NADH were detected with LC/MS (n = 6). * P < 0.05, ** P < 0.01.

Fig.7 SIRT5 plays an important role in ketogenesis of SB. (A) Protein succinylation of primary hepatocytes of C57BL/6 mice in the presence of SIRT5 inhibitor MC3482. The cells were pretreated with MC3482 (100 μmol/L) for 1 h before the SB (2.5 mmol/L) treatment. (B) Quantification of A (n = 4). (C) BHB in the supernatant of primary hepatocytes treated with MC3482 and SB (2.5 mmol/L) (n = 6). (D) SIRT5 and HMGCS2 proteins in the liver of Sirt5-KO mice. (E) Protein succinylation in the liver of Sirt5-KO mice. (F) Succinylation of HMGCS2 protein in the liver of Sirt5-KO mice in the IP study. (G) Blood ketone body levels in the Sirt5-KO mice in the fasting and non-fasting conditions (WT, n = 7; Sirt5-KO, n = 6). (H) Blood BHB levels in Sirt5-KO mice with SB challenge (2.5 g/kg, i.p.) for 4 h (n = 6). (I) BHB in the supernatant of primary hepatocytes of Sirt5-KO mice (n = 6). * P < 0.05, ** P < 0.01, *** P < 0.001.

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