Stearoyl-CoA desaturase 1 deficiency increases fatty acid oxidation by activating AMP-activated protein kinase in liver

Abstract

Stearoyl-CoA desaturase (SCD) catalyzes the rate-limiting step in the biosynthesis of monounsaturated fatty acids. Mice with a targeted disruption of the SCD1 isoform have reduced body adiposity, increased energy expenditure, and up-regulated expression of several genes encoding enzymes of fatty acid beta-oxidation in liver. The mechanisms by which SCD deficiency leads to these metabolic changes are presently unknown. Here we show that the phosphorylation and activity of AMP-activated protein kinase (AMPK), a metabolic sensor that regulates lipid metabolism during increased energy expenditure is significantly increased (approximately 40%, P < 0.01) in liver of SCD1 knockout mice (SCD1-/-). In parallel with the activation of AMPK, the phosphorylation of acetyl-CoA carboxylase at Ser-79 was increased and enzymatic activity was decreased (approximately 35%, P < 0.001), resulting in decreased intracellular levels of malonyl-CoA (approximately 47%, P < 0.001). An SCD1 mutation also increased AMPK phosphorylation and activity and increased acetyl-CoA carboxylase phosphorylation in leptin-deficient ob/ob mice. Lower malonyl-CoA concentrations are known to derepress carnitine palmitoyltransferase 1 (CPT1). In SCD1-/- mice, CPT1 and CPT2 activities were significantly increased (in both cases approximately 60%, P < 0.001) thereby stimulating the oxidation of mitochondrial palmitoyl-CoA. Our results identify AMPK as a mediator of increased fatty acid oxidation in liver of SCD1-deficient mice.

Fig. 1.

AMPK is activated in SCD1-/- mice. (A) Phospho-AMPK and α1 and α2 AMPK protein subunits were quantified by using the Western blot technique. (B) The AMPK activity in PEG 8000 fraction, with SAMS peptide as substrate, was assessed as described in Materials and Methods. **, P < 0.01 vs. control.

Fig. 2.

Phospho-ACC and ACC protein level (A), ACC activity (B), and malonyl-CoA concentration (C) in liver of SCD1+/+ and SCD1-/- mice. Phospho-ACC [visible are the two known ACC isoforms, ACC1 (265 kDa) and ACC2 (280 kDa)], ACC1, and ACC2 proteins were quantified by Western blotting. ACC activity in the 6% PEG 8000 fraction was determined by using the [¹⁴C]bicarbonate fixation assay. Reaction was conducted for 2 min in the presence of 10 mM citrate and stopped by adding 10% perchloric acid. Malonyl-CoA level was measured radiochemically according to McGarry et al. (29). Highly purified fatty acid synthetase from chicken was used. ***, P < 0.001 vs. controls.

Fig. 3.

CPT1 protein level (A), CPT1 and CPT2 activities (B), and effect of malonyl-CoA on CPT1 activity (C) in mitochondria of SCD1+/+ and SCD1-/- mice. CPT1 protein level was measured by the Western blot technique. Intact mitochondria were separated according to Vance (21), and CPT1 and CPT2 activities were measured radiochemically by using l-[³H]carnitine. CPT2 activity was measured in Tween 20 extracts of mitochondria as described in Materials and Methods. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 vs. controls.

Fig. 4.

Incorporation of [¹⁴C]palmitic acid into lipids (A) and palmitoyl-CoA oxidation in mitochondria of SCD1+/+ and SCD1-/- mice (B). Ten microcurie of [¹⁴C]palmitic acid was administered in the tail vein. Ten minutes after administration of the label, liver was taken and homogenized. Then lipids were extracted and assayed for radioactivity in a liquid scintillation counter. Oxidation of palmitoyl-CoA was measured in isolated mitochondria and presented as the sum of labeled CO₂ released during oxidation, [¹⁴C] content in the ketone bodies fraction, and acid-soluble labeled oxidation products as described in Materials and Methods. **, P < 0.01; and ***, P < 0.001 vs. controls.

Fig. 5.

SCD1 deficiency increases AMPK phosphorylation (A) and activity (B) and ACC phosphorylation (C) in ob/ob mice. Phospho-AMPK and α1 and α2 AMPK protein subunits in liver of ob/ob (ab^J/ab⁺;ob/ob) and double-mutant (ab^J/ab^J;ob/ob) mice were assayed by using the Western blot technique. AMPK activity was assayed as described in Materials and Methods. Phospho-ACC1 (265 kDa) and ACC2 (280 kDa) isoforms and ACC1 and ACC2 protein levels were assayed by Western blotting. *, P < 0.05 vs. control.

Fig. 6.

Changes in lipid metabolism due to activation of AMPK in liver of mice with disruption of SCD1 gene: a proposed model. Activation of AMPK decreases ACC activity that in turn decreases malonyl-CoA level in liver. A drop in malonyl-CoA content activates CPT1 activity and increases transport of fatty acids into mitochondria for β-oxidation. AMPK can also phosphorylate a cytosolic protein that activates CPT1 (33).?, Mechanism of AMPK activation by SCD1 deficiency is unknown.