Regulation of UCP1 and Mitochondrial Metabolism in Brown Adipose Tissue by Reversible Succinylation

Abstract

Brown adipose tissue (BAT) is rich in mitochondria and plays important roles in energy expenditure, thermogenesis, and glucose homeostasis. We find that levels of mitochondrial protein succinylation and malonylation are high in BAT and subject to physiological and genetic regulation. BAT-specific deletion of Sirt5, a mitochondrial desuccinylase and demalonylase, results in dramatic increases in global protein succinylation and malonylation. Mass spectrometry-based quantification of succinylation reveals that Sirt5 regulates the key thermogenic protein in BAT, UCP1. Mutation of the two succinylated lysines in UCP1 to acyl-mimetic glutamine and glutamic acid significantly decreases its stability and activity. The reduced function of UCP1 and other proteins in Sirt5KO BAT results in impaired mitochondria respiration, defective mitophagy, and metabolic inflexibility. Thus, succinylation of UCP1 and other mitochondrial proteins plays an important role in BAT and in regulation of energy homeostasis.

Keywords: UCP1; brown fat; mitochondria; succinylation; thermogenesis.

Figure 1.. Protein acylation level is high in BAT and subject to physiological and genetic regulation.

Protein acylation and Sirt5 expression were assessed by Western blotting. (A)Total tissue lysates of BAT, sWAT, eWAT and liver from random-fed, 9 week-old wild type C57/B6J mice; (B) BAT from 13–14 week-old db/+ vs. db/db mice; (C) BAT from wild type mice fed high fat diet (60% fat) for 12 weeks and aged matched chow-fed mice; (D) BAT from wild type mice housed at 22°C, or acclimated to 5°C or 30°C for 10 days, and from wild type mice acutely exposed to 6°C after acclimation at 30°C for 10 days; (E) Total cell lysates and mitochondrial fractions of BAT from mice housed at 22°C or acclimated to 5°C for 10 days. n=4 to 6 per group. White dashed lines were used to separate groups in a single gel

Figure 2.. BAT specific-Sirt5 deficiency elevates protein succinylation and malonylation levels.

(A) H&E staining of BAT from 11 week-old random-fed floxed and 5-BKO mice, scale bar=100 μm (B) Ratio of adipose tissue weight vs. body weight in 11 week-old chow fed mice, n=5 vs. 6. Data are represented as mean ± SEM. (C) Q-PCR of BAT from 11 week-old random-fed floxed and 5-BKO mice, n=6 per group. Data are represented as mean ± SEM, *p < 0.05, student’s t-test. (D) Western blot of BAT from 2 month-old female floxed and 5-BKO mice housed at 22°C or acclimated to 5°C for 10 days. (E) Western blot of BAT cytoplasmic (cyto) and mitochondrial fraction (mito) from 8 week-old, 24 hr-fasted floxed and 5-BKO mice.

Figure 3.. Sirt5 deficiency leads to metabolic inflexibility in BAT.

(A) Rectal temperature of 2 month-old floxed and 5-BKO mice during acute cold (from 22°C to 7°C) exposure. Mice were allowed free access to food and water. n=5 vs. 7. Data are represented as mean ± SEM. (B) Rectal temperature of 2.5 month-old floxed and 5-BKO mice during acute cold (from 22°C to 7°C) exposure. Mice were fasted for 18 hours. n=7 vs. 8. Data are represented as mean ± SEM. (C) Intra-abdominal temperature of HFD fed floxed and Sirt5-BKO mice after 3 days’ acclimation to 5°C, recorded at 15 min interval. Dark phase are shaded, measurements occurred between 10:00 p.m. and 2:00 a.m. were boxed. n=4 vs. 6. Data are represented as mean ± SEM. *p < 0.05. (D) Respiration exchange ratio (RER) of 5 month-old floxed and 5-BKO mice on chow diet. n=6. Data are represented as mean ± SEM. (E) Glucose tolerance test of floxed and 5-BKO mice fed with high fat (60% by calories) diet for 11 weeks. n=5 vs. 8. Data are represented as mean ± SEM. (F) Insulin tolerance tests of floxed and 5-BKO mice fed with high fat diet for 12 weeks. n=6 vs. 8, Data are represented as mean ± SEM. (F) Fasting plasma insulin levels of floxed and 5-BKO mice fed with high fat diet for 13 weeks. n=6 vs. 8. Data are represented as mean ± SEM. *p < 0.05, student’s t-test.

Figure 4.. BAT Sirt5 desuccinylates proteins in major metabolic pathways.

(A) Venn diagram showing lysine sites whose succinylation levels were increased (red) or decreased (blue) by at least two fold with FDR<0.01 comparing between Sirt5 KO vs. Floxed and cold acclimation (CA) vs. room temperature (RT) housed mice. (B) Reactome term enrichment analysis from the list of proteins containing succinylation sites that were identified by mass spectrometry and increased due to Sirt5 KO. (C-F) Succinylation fold changes (F.C.) on individual lysine sites of SDHA (C), SDHB (D), GLUD1 (E), and UCP1 (F). (G) Western blot of BAT from floxed and 5-BKO mice before and after anti-succinyl-K immunoprecipitation.

Figure 5.. Hypersuccinylation due to Sirt5 deficiency impairs mitochondria respiration and enzymatic activity.

(A-C) Oxygen consumption rate (OCR) in Seahorse flux assay using isolated mitochondria of BAT from overnight fasted floxed and 5-BKO mice. n=10 wells from 5 mice per genotype. Data are represented as mean ± SEM. TMPD: N, N, N′, N′-tetramethyl-p-phenylenediamine (D) Succinate and (E) fumarate concentrations in 50% aqueous acetonitrile homogenates of BAT from random fed floxed and 5-BKO mice. n=5 vs. 6. Data are represented as mean ± SEM. (F) Succinate dehydrogenase (SDH) activity in BAT mitochondria isolated from floxed and 5-BKO mice. n=7 vs. 5. Data are represented as mean ± SEM. (G) Oil Red O staining of day 6 mature brown adipocytes. (H) Western blot of day 6 mature brown adipocytes. (I) Fatty acid oxidation assay using C¹⁴-palmitic acid in day 7 mature floxed and Sirt5 KO brown adipocytes. n=6. Data are represented as mean ± SD. ASM: acid soluble metabolite. (J) Oxygen consumption rate (OCR) in Seahorse flux assay using isolated mitochondria of BAT from overnight fasted female floxed and 5-BKO mice. n=10 wells from 5 pools (2 mice per pool) per genotype. Data are represented as mean ± SEM. (K) Glutamate oxidation assay and (L) glutamate uptake assay in the presence or absence of leucine in day 7 mature floxed and Sirt5 KO brown adipocytes. n=6. Data are represented as mean ± SD. *p < 0.05, student’s t-test with Bonferroni correction. (M) OCR and (N) ECAR of Seahorse flux assay in day 5 floxed and Sirt5 KO brown adipocytes. n=10 wells per group, normalized to protein amount. Data are represented as mean ± SEM. Glc: glucose. 2-DG: 2-deoxy-glucose. Eto: etomoxir. R/A: rotenone+ antimycin, *p < 0.05, student’s t-test.

Figure 6.. Overacylation due to sirt5 deficiency impairs UCP1 activity and stability.

(A) Mouse UCP1 structure model showing location of K56 and K151. (B) UCP1 mRNA expression in confluent 3T3-L1 pre-adipocytes stably infected with retrovirus expressing MSCV-puro vector, or wildtype or 2KQ mutant of UCP1. n=4 in each group. Data are represented as mean ± SD. (C) UCP1 protein expression in confluent 3T3-L1 preadipocytes as in Figure 6B. (D) Seahorse Flux assay using confluent 3T3-L1 preadipocytes transduced as in Figure 6B. Oligo: oligomycin, R/A: rotenone+antimycin. (E) Quantitation of OCR for Seahorse in Figure 6D. n=6 for vector, n=7 for WT and 2KQ UCP1. Data are represented as mean ± SD. *p < 0.05, Student’s t-test with Bonferroni correction. (F) UCP1 mRNA expression in confluent 3T3-L1 pre-adipocytes stably infected with retrovirus expressing MSCV-puro vector, or wildtype or 2KE mutant of UCP1. n=4 in each group. Data are represented as mean ± SD. (G) UCP1 protein expression in confluent 3T3-L1 pre-adipocytes as in Figure 6F. (H) Seahorse Flux assay using confluent 3T3-L1 pre-adipocytes transduced as in Figure 6F. (I) Quantitation of OCR for Seahorse in Figure 6H. n=6 for vector, n=7 for WT and 2KE UCP1. Data are represented as mean ± SD. *p < 0.05, Student’s t-test with Bonferroni correction. (J) Western blot of 3T3-L1 preadipocytes transduced with WT-UCP1 or the 2KQ/2KE mutant and treated with 20 μg/mL cycloheximide (CHX) for 0, 3.5 and 7 hrs. Global ubiquitination were used as positive control for CHX effect.

Figure 7.. Overacylation due to Sirt5 deficiency leads to autophagy/mitophagy defect.

(A) Western blot of selected mitochordrial and autophagy/mitophagy markers in BAT extracts from 2.5-month-old random fed and 24 h fasted floxed and 5-BKO mice. n=4 per group. (B) Quantitation of the 24 h fasted group in Fig.7A. n=4. Data are represented as mean ± SEM. *p < 0.05, student’s t-test. (C) Q-PCR analysis of Mito Oxphos genes in BAT from 2.5-month-old random fed (same mice as Figure 2C) and 24 h fasted floxed and 5-BKO mice. n=5 to 6 per group. Data are represented as mean ± SEM. *p < 0.05, student’s t-test. (D) Q-PCR analysis of autophagy and mitophagy genes in BAT as in Fig.7C. (E) Histogram of BAT mitochondria size quantification from 24 hour fasted chow fed 2-month-old floxed and 5-BKO mice. Electron microscopic (EM) images of 600 mitochondria from 4 mice per genotype were quantified using image J. Data are represented as mean ± SEM. *p < 0.05, student’s t-test. (F) Representative EM pictures showing mitochondria size and morphology from each genotype, scale bar =500 nm. (G) Western blot of BAT from 2-month-old floxed and 5-BKO mice 3 hours after saline or leupeptin (40mg/kg) injection following 21 hour fast. n=3. (H) Quantification of LC3II protein amount in Figure 7G, normalized to Vinculin. Data are represented as mean ± SEM. *p < 0.05, student’s t-test.