Rapid nitration of adipocyte phosphoenolpyruvate carboxykinase by leptin reduces glyceroneogenesis and induces fatty acid release

Abstract

Fatty acid (FA) release from white adipose tissue (WAT) is the result of the balance between triglyceride breakdown and FA re-esterification. The latter relies on the induction of cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C), the key enzyme for glyceroneogenesis. We previously demonstrated that long-term (18 h) leptin treatment of rat epididymal WAT explants reduced glyceroneogenesis through nitric oxide (NO)-induced decrease in PEPCK-C expression. We investigated the effect of a short-term leptin treatment (2 h) on PEPCK-C expression and glyceroneogenesis in relation to NO production. We demonstrate that in WAT explants, leptin-induced NO synthase III (NOS III) phosphorylation was associated with reduced PEPCK-C level and glyceroneogenesis, leading to FA release, while PEPCK-C gene expression remained unaffected. These effects were absent in WAT explants from leptin receptor-deficient Zucker rat. Immunoprecipitation and western blot experiments showed that the leptin-induced decrease in PEPCK-C level was correlated with an increase in PEPCK-C nitration. All these effects were abolished by the NOS inhibitor Nω-nitro-L-arginine methyl ester and mimicked by the NO donor S-nitroso-N-acetyl-DL penicillamine. We propose a mechanism in which leptin activates NOS III and induces NO that nitrates PEPCK-C to reduce its level and glyceroneogenesis, therefore limiting FA re-esterification in WAT.

Figure 1. NO-dependent effects of leptin on glycerol (A) and FA (B) release from rat WAT explants.

Explants were pre-treated or not with L-NAME (1 mmol/L) for 30 min, then exposed or not to either leptin (10 µg/L), SNAP (1 mmol/L) or IFN-γ (50 µg/L) for 2 h in KRB medium containing 2% BSA (lipolysis medium). Results are expressed as the percent of glycerol or FA relative to the corresponding untreated control. Crude values for control were 4.57±0.13 nmol.mg⁻¹ tissue.2 h⁻¹ for glycerol and 4.60±0.47 nmol.mg⁻¹ tissue. 2 h⁻¹ for FA. Each value represents the mean ± SEM, (n = 4) *, P<0.01 vs. control; ^a P<0.01 vs. leptin-treated explants.

Figure 2. NO-dependent effects of leptin on glyceroneogenesis and NOS III phosphorylation in WAT explants from rats.

Explants from SD (A) or Zucker rats (B, C) were pre-treated or not with L-NAME (1 mmol/L) or AG490 (10 µmol/L) for 30 min, then exposed or not to either leptin (10 µg/L), SNAP (1 mmol/L) or IFN-γ (50 µg/L) for 2 h in KRB medium containing 2% BSA and [1-¹⁴C]-pyruvate. Glyceroneogenic flux was measured by the [1-¹⁴C]-pyruvate incorporation into neutral lipids. Each value represents the mean ± SEM, (n = 4) *, P<0.01 vs. control; ^a P<0.01 vs. leptin-treated explants. (C) Representative autoradiogram of a western blot performed on WAT cytosolic proteins from Zucker rats reveals total NOS III and its Ser¹¹⁷⁹ phosphorylated form.

Figure 3. Effects of leptin and IFN-γ on PEPCK-C, NOS II and NOS III mRNA in rat WAT explants.

Explants were treated or not with leptin (10 µg/L) or IFN-γ (50 µg/L) for 2 h. PEPCK-C, NOS II and NOS III mRNA levels were analysed by RT-qPCR. Results are normalized using 18S rRNA. Each value represents the mean ± SEM, (n = 4).

Figure 4. NO-dependent effects of leptin on PEPCK-C protein in WAT explants from rats.

WAT explants were pre-treated or not with L-NAME (1 mmol/L) for 30 min, then exposed or not to either leptin (10 µg/L), SNAP (1 mmol/L) or INF-γ (50 µg/L) for 2 h in KRB medium containing 2% BSA. PEPCK-C and β-actin proteins were revealed by western blotting performed on cytosolic proteins. (A) Densitometry scanning in ImageJ software. Each value represents the mean ± SEM (n = 4) *, P<0.01 vs. control; ^a P<0.01 vs. leptin-treated explants. (B) Representative autoradiograms.

Figure 5. NO-dependent effect of leptin on the nitration of PEPCK-C protein in WAT explants from rats.

WAT explants were pre-treated or not with L-NAME (1 mmol/L) for 30 min, then exposed or not to either leptin (10 µg/L), SNAP (1 mmol/L) or IFN-γ (50 µg/L) for 2 h in KRB medium containing 2% BSA. We first immunoprecipited cytosolic proteins using the anti-PEPCK-C antibody then proceeded to western blotting with the anti-nitroprotein antibody. (A) Densitometry scanning in ImageJ software. Each value represents the mean ± SEM, (n = 4) *, P<0.01 vs. control; ^a P<0.01 vs. leptin-treated explants. (B) Representative autoradiogram.