Diabetes Metab J.  2023 May;47(3):382-393. 10.4093/dmj.2022.0156.

Role of SUMO-Specific Protease 2 in Leptin-Induced Fatty Acid Metabolism in White Adipocytes

• Kim PC
• Lee JS
• Chung SS
• Park KS

• KMID: 2542515
• DOI: http://doi.org/10.4093/dmj.2022.0156

Abstract

Background
Leptin is a 16-kDa fat-derived hormone with a primary role in controlling adipose tissue levels. Leptin increases fatty acid oxidation (FAO) acutely through adenosine monophosphate-activated protein kinase (AMPK) and on delay through the SUMO-specific protease 2 (SENP2)–peroxisome proliferator-activated receptor δ/γ (PPARδ/γ) pathway in skeletal muscle. Leptin also directly increases FAO and decreases lipogenesis in adipocytes; however, the mechanism behind these effects remains unknown. Here, we investigated the role of SENP2 in the regulation of fatty acid metabolism by leptin in adipocytes and white adipose tissues.
Methods
The effects of leptin mediated by SENP2 on fatty acid metabolism were tested by siRNA-mediated knockdown in 3T3-L1 adipocytes. The role of SENP2 was confirmed in vivo using adipocyte-specific Senp2 knockout (Senp2-aKO) mice. We revealed the molecular mechanism involved in the leptin-induced transcriptional regulation of carnitine palmitoyl transferase 1b (Cpt1b) and long-chain acyl-coenzyme A synthetase 1 (Acsl1) using transfection/reporter assays and chromatin immunoprecipitation.
Results
SENP2 mediated the increased expression of FAO-associated enzymes, CPT1b and ACSL1, which peaked 24 hours after leptin treatment in adipocytes. In contrast, leptin stimulated FAO through AMPK during the initial several hours after treatment. In white adipose tissues, FAO and mRNA levels of Cpt1b and Acsl1 were increased by 2-fold 24 hours after leptin injection in control mice but not in Senp2-aKO mice. Leptin increased PPARα binding to the Cpt1b and Acsl1 promoters in adipocytes through SENP2.
Conclusion
These results suggest that the SENP2-PPARα pathway plays an important role in leptin-induced FAO in white adipocytes.

Keyword

Adipocytes; white; Fatty acids; Leptin; Peroxisome proliferator-activated receptors; Senp2 protein; mouse

Figure

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  Fig. 1 Leptin increased fatty acid oxidation (FAO) acutely via adenosine monophosphate-activated protein kinase (AMPK) in 3T3-L1 adipocytes. (A) Relative leptin receptor b (Leprb) mRNA levels of several types of fat, brown adipose tissue (BAT), subcutaneous adipose tissue (SAT), visceral adipose tissue (VAT), hypothalamus (Hypothal), soleus muscle, and liver from wild type C57BL/6J mice. The mRNA levels of Leprb were expressed as ΔCt (Leprb Ct - glyceraldehyde-3-phosphate dehydrogenase [Gapdh] Ct). Data are presented as mean±standard error of the mean (SEM) of four different mice. (B) The Leprb mRNA levels of C2C12 myotubes and 3T3-L1 adipocytes were measured and expressed as ΔCt (Leprb Ct - Gapdh Ct). Data are presented as mean±SEM of five to eight different cell preps. (C, D) After pre-treatment with compound C (10 μM) for 1 hour, 3T3-L1 adipocytes were treated with leptin (50 ng/mL) for 6 hours (C) or 24 hours (D), and then FAO was measured. FAO levels were expressed as dpm/μg of proteins (n=3). All data are presented as mean±SEM. n, number of individual experiments. aP<0.05 vs. vehicle, bP<0.05 vs. leptin without compound C.

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  Fig. 2 Leptin increased SUMO-specific protease 2 (Senp2) expression via signal transducer and activator of transcription 3 (STAT3) in 3T3-L1 adipocytes. (A) After serum starvation for 24 hours, 3T3-L1 adipocytes were treated with leptin at different concentrations (10, 25, or 50 ng/mL) for 3 hours. Cell lysates were subjected to Western blotting using antibodies against phospho-STAT3 (pSTAT3) and total STAT3 (STAT3). (B) After serum starvation for 24 hours, 3T3-L1 adipocytes were treated with leptin (50 ng/mL) for 3, 6, 10, or 24 hours. Real-time quantitative polymerase chain reaction analysis was performed with Senp2 primers. The mRNA level of vehicle was expressed as 1, and the others were expressed as its relative values (n=5). (C) After nonspecific small interfering RNA (siNS) or siRNA against Stat3 (siStat3) (100 nM) treatment for 48 hours, 3T3-L1 adipocytes were treated with leptin (50 ng/mL) for 24 hours. The Senp2 mRNA level of siNS without leptin treatment was expressed as 1, and the others were expressed as its relative values (n=3). All data are presented as mean±standard error of the mean. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; a.u., arbitrary unit. aP<0.05 vs. 0 hour, bP<0.05 vs. vehicle, cP<0.05 vs. siNS/leptin.

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  Fig. 3 Leptin increased expressions of fatty acid oxidation (FAO)-associated enzymes through SUMO-specific protease 2 (SENP2) in 3T3-L1 adipocytes. (A) After serum starvation for 24 hours, 3T3-L1 adipocytes were treated with leptin (50 ng/mL), and real-time quantitative polymerase chain reaction (RT-qPCR) analysis was performed using primers specific to carnitine palmitoyl transferase 1b (Cpt1b, left) or long-chain acyl-coenzyme A synthetase 1 (Acsl1, right). The mRNA level at 0 hour was expressed as 1, and the others were expressed as its relative values (n=5). (B, C) After nonspecific small interfering RNA (siNS) or siRNA against Senp2 (siSenp2, 200 nM) treatment for 48 hours, 3T3-L1 adipocytes were treated with leptin for 24 hours, and then RT-qPCR analysis was performed using primers for Senp2 (B), Cpt1b and Acsl1 (C). The mRNA levels of siNS without leptin treatment were expressed as 1, and the others were expressed as their relative values (n=3). (D, E) 3T3-L1 adipocytes were transfected with siNS or siSenp2 for 48 hours followed by leptin treatment for 6 hours (D) or 24 hours (E), and then FAO levels of the adipocytes were measured (n=3). All data are presented as mean±standard error of the mean. a.u., arbitrary unit. aP<0.05 vs. 0 hour, bP<0.05 vs. siNS/vehicle, cP<0.05 vs. siNS/leptin.

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  Fig. 4 Leptin increased fatty acid oxidation (FAO) in white adipose tissues in vivo via SUMO-specific protease 2 (SENP2). (A, B) Ten weeks old control and adipocyte-specific Senp2 knockout (Senp2-aKO) mice were injected with leptin (3 mg/kg) or saline intraperitoneally. Mice tissues, including visceral adipose tissue (VAT), subcutaneous adipose tissue (SAT), and soleus muscle (Soleus), were obtained 24 hours after the injection, and FAO (A) or mRNA levels of Senp2, carnitine palmitoyl transferase 1b (Cpt1b), and long-chain acyl-coenzyme A synthetase 1 (Acsl1) (B) were measured. (A) FAO was expressed as dpm/μg of proteins (n=3 mice). (B) The mRNA level of each tissue from saline-injected control mice was expressed as 1, and the others were expressed as its relative values (n=3 mice). All data are presented as mean±standard error of the mean. a.u., arbitrary unit. aP<0.05 vs. control/saline, bP<0.05 vs. control/leptin.

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  Fig. 5 SUMO-specific protease 2 (SENP2) increased binding of peroxisome proliferator-activated receptor α (PPARα) on PPAR response element (PPRE) sites in the carnitine palmitoyl transferase 1b (Cpt1b) and long-chain acyl-coenzyme A synthetase 1 (Acsl1) promoters upon leptin treatment in 3T3-L1 adipocytes. (A, B) 3T3-L1 preadipocytes were transfected with 300 ng of the Cpt1b promoter-luc constructs (A), and the Acsl1 promoter-luc constructs (B), followed by leptin treatment (50 ng/mL) for 24 hours. Luciferase activity of the cells transfected with mCpt1b(−320)-luc or mAcsl1(−1,051)-luc without leptin treatment was expressed as 100, and the others were expressed as their relative values (n=5). (C, D) After nonspecific small interfering RNA (siNS) or siRNA against Senp2 (siSenp2, 200 nM) treatment for 48 hours, 3T3-L1 adipocytes were treated with leptin for 24 hours, and then subjected to chromatin immunoprecipitation (ChIP)-coupled quantitative polymerase chain reaction analysis using antibodies against PPARα, PPARδ, and PPARγ and the Cpt1b PPRE primers (C) or Acsl1 PPRE primers (D). Binding activity was expressed as its % input (n=3). (E, F) After siNS, siPparα, siPparδ, or siPparγ (100 nM) treatment for 48 hours, 3T3-L1 adipocytes were treated with leptin for 24 hours, and then subjected to real-time quantitative polymerase chain reaction analysis using Cpt1b primers (E) or Acsl1 primers (F). The mRNA levels of siNS/vehicle were expressed as 1, and the others were expressed as their relative values (n=4). All data are presented as mean±standard error of the mean. a.u., arbitrary unit. aP<0.05 vs. vehicle, bP<0.05 vs. siNS/leptin.

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