LKB1 regulates lipid oxidation during exercise independently of AMPK

Abstract

Lipid metabolism is important for health and insulin action, yet the fundamental process of regulating lipid metabolism during muscle contraction is incompletely understood. Here, we show that liver kinase B1 (LKB1) muscle-specific knockout (LKB1 MKO) mice display decreased fatty acid (FA) oxidation during treadmill exercise. LKB1 MKO mice also show decreased muscle SIK3 activity, increased histone deacetylase 4 expression, decreased NAD⁺ concentration and SIRT1 activity, and decreased expression of genes involved in FA oxidation. In AMP-activated protein kinase (AMPK)α2 KO mice, substrate use was similar to that in WT mice, which excluded that decreased FA oxidation in LKB1 MKO mice was due to decreased AMPKα2 activity. Additionally, LKB1 MKO muscle demonstrated decreased FA oxidation in vitro. A markedly decreased phosphorylation of TBC1D1, a proposed regulator of FA transport, and a low CoA content could contribute to the low FA oxidation in LKB1 MKO. LKB1 deficiency did not reduce muscle glucose uptake or oxidation during exercise in vivo, excluding a general impairment of substrate use during exercise in LKB1 MKO mice. Our findings demonstrate that LKB1 is a novel molecular regulator of major importance for FA oxidation but not glucose uptake in muscle during exercise.

FIG. 1.

LKB1 MKO mice are exercise intolerant and display reduced oxygen uptake and limited FA oxidation during exercise in vivo. A: Maximal-speed running test of LKB1 MKO and WT mice was performed. B and C: RER (B) and oxygen uptake (C) (mL ⋅ h⁻¹ ⋅ kg⁻¹) during treadmill exercise at 60% of maximal running speed and at the same absolute intensity. Whole-body FA oxidation (kJ ⋅ h⁻¹ ⋅ kg⁻¹) (D) and carbohydrate (CHO) oxidation (kJ ⋅ h⁻¹ ⋅ kg⁻¹) (E) were calculated from RER values as described in research design and methods. Data are presented as means ± SEM (n = 11–12). **P < 0.01, ***P < 0.001, significantly different from WT; #P < 0.05, significant difference between WT groups.

FIG. 2.

LKB1 signaling in LKB1 MKO and WT mice during in vivo exercise. A: LKB1 protein was almost completely ablated in LKB1 MKO mice. B–E: LKB1 activity and activity of downstream targets were determined in gastrocnemius muscle from LKB1 MKO and WT mice. F and G: AMPK Thr172 and ACC2 Ser212 phosphorylation (phos) in gastrocnemius muscle from LKB1 MKO and WT mice. Malonyl-CoA (H) and free CoA (I) content in gastrocnemius muscle in WT and LKB1 MKO mice. Data are presented as means ± SEM (n = 6–13). *P < 0.05, **P < 0.01, ***P < 0.001, significantly different from WT; #P = 0.05, significant effect of exercise, ##P < 0.01, significant difference between resting and exercising WT mice. Ex; exercise, exp; protein expression, ww; wet weight.

FIG. 3.

Lack of LKB1 reduces the capacity for FA oxidation in isolated EDL muscle. A: FA oxidation was determined at rest and after contractions in EDL muscle from LKB1 MKO and WT mice. B: AMPK Thr172 and ACC2 phosphorylation (phos) in resting and contracted EDL muscle from LKB1 MKO and WT mice. Data are presented as means ± SEM (n = 11–12). *P < 0.05, ***P < 0.001, significantly different from WT. #P < 0.05, ###P < 0.001, significant difference between WT groups.

FIG. 4.

LKB1-deficient muscle has lower SIRT1 activity, increased HDAC4 expression (exp), and reduced FA oxidative gene expression. SIRT1 protein (A), NAD⁺ concentration (B), and the SIRT1 substrate acetylated p53 Lys379 (C) were determined in gastrocnemius muscle. HDAC4 protein expression (D) and phosphorylated (phos) HDAC4 Ser632 (E) were determined in gastrocnemius muscle from LKB1 MKO and WT mice. β-Tubulin, which was similar between genotypes, was used as loading control for HDAC4 expression. F: Heat map of 27 genes found to be differentially expressed between WT and LKB1 MKO (n = 4) muscle biopsies and involved in lipid metabolism. The fold change in gene expression is color coded: red, upregulation; blue, downregulation. Based on their expression, biopsies are separated in two distinct clusters: one containing WT muscles and the other LKB1 MKO muscles. The dendrogram above the heat map displays hierarchical clustering of the columns based on a distances matrix where the height of lines indicates the degree of separation between clusters. Data are presented as means ± SEM (n = 7–12). *P < 0.05, **P < 0.01, significantly different from WT. AU, arbitrary units.

FIG. 5.

Skeletal muscle deficient in LKB1 has reduced mitochondrial enzyme activity but displays normal mitochondrial network and ultrastructural morphology. Citrate synthase (CS) activity (A) and HAD activity (B) (n = 9–10) in gastrocnemius muscle from LKB1 MKO and WT mice. Data are presented as means ± SEM. *P < 0.05, significantly different from WT. C: Mitochondrial network in single muscle fibers from WT and LKB1 MKO mice visualized using immunocytochemical staining of COXIV and imaged by confocal immunofluorescence microscopy (n = 3); bar = 10 μm. D: TEM on cross-sections from quadriceps muscle from WT and LKB1 MKO mice was performed. Left panel: A stitched image combined of 100 individual images (10 × 10); bar = 10 μm. Middle panel: Representative images of subsarcolemmal (SS) mitochondria; bar = 1 μm. Right panel: Representative images of intermyofibrillar (IMF) mitochondria; bar = 1 μm.

FIG. 6.

LKB1 MKO mice display normal increases in muscle glucose clearance in response to treadmill exercise. 2-deoxyglucose (2DG) clearance (mL ⋅ g⁻¹ ⋅ min⁻¹) was determined in quadriceps (A) and EDL muscle (B) from LKB1 MKO and WT mice at rest and in response to treadmill exercise at the same relative intensity. Muscle glycogen (C) (µmol ⋅ g wet wt⁻¹) and muscle lactate content (D) in quadriceps muscle from WT and LKB1 MKO mice at rest and in response to 20 min treadmill running exercise at the same relative intensity. Data are presented as means ± SEM (n = 6–13). #P < 0.05, ##P < 0.01, significantly different from rest within genotype. ND, not determined.

FIG. 7.

LKB1 MKO mice display reduced glucose uptake during intense, but not moderate, muscle contractions ex vivo. A: 2-deoxyglucose (2DG) uptake (µmol ⋅ g⁻¹ ⋅ h⁻¹) in EDL muscle from WT and LKB1 MKO mice at basal and in response to 10 min moderate electrical stimulation (n = 9–11). B: 2-deoxyglucose uptake (µmol ⋅ g⁻¹ ⋅ h⁻¹) in EDL muscle from LKB1 MKO and WT mice subjected to intense electrically stimulated muscle contraction for 10 min (n = 6–9). C: TBC1D1 Ser237 phosphorylation (phos) in EDL muscles in response to moderate and intense electrically induced muscle contraction (n = 7–11). Data are presented as means ± SEM. *P < 0.05, **P < 0.01, significantly different from WT. #P < 0.05, ##P < 0.01, significantly different from rest.