TLR9 and beclin 1 crosstalk regulates muscle AMPK activation in exercise

Abstract

The activation of adenosine monophosphate-activated protein kinase (AMPK) in skeletal muscle coordinates systemic metabolic responses to exercise1. Autophagy—a lysosomal degradation pathway that maintains cellular homeostasis2—is upregulated during exercise, and a core autophagy protein, beclin 1, is required for AMPK activation in skeletal muscle3. Here we describe a role for the innate immune-sensing molecule Toll-like receptor 9 (TLR9)4, and its interaction with beclin 1, in exercise-induced activation of AMPK in skeletal muscle. Mice that lack TLR9 are deficient in both exercise-induced activation of AMPK and plasma membrane localization of the GLUT4 glucose transporter in skeletal muscle, but are not deficient in autophagy. TLR9 binds beclin 1, and this interaction is increased by energy stress (glucose starvation and endurance exercise) and decreased by a BCL2 mutation3,5 that blocks the disruption of BCL2–beclin 1 binding. TLR9 regulates the assembly of the endolysosomal phosphatidylinositol 3-kinase complex (PI3KC3-C2)—which contains beclin 1 and UVRAG—in skeletal muscle during exercise, and knockout of beclin 1 or UVRAG inhibits the cellular AMPK activation induced by glucose starvation. Moreover, TLR9 functions in a muscle-autonomous fashion in ex vivo contraction-induced AMPK activation, glucose uptake and beclin 1–UVRAG complex assembly. These findings reveal a heretofore undescribed role for a Toll-like receptor in skeletal-muscle AMPK activation and glucose metabolism during exercise, as well as unexpected crosstalk between this innate immune sensor and autophagy proteins.

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Fig. 1: TLR9 interacts with beclin 1 during glucose starvation and exercise.
Fig. 2: TLR9 is required for exercise-induced activation of AMPK in muscles.
Fig. 3: TLR9 is required for the beclin 1–UVRAG interaction during exercise.

Data availability

Full scans for all western blots are provided in Supplementary Fig. 1. Source Data for Figs. 13 and Extended Data Figs. 110 are provided with the paper. All other data are available from the corresponding author on reasonable request.

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Acknowledgements

We thank R. Bassel-Duby, C. He, R. Medzhitov and P. Scherer for helpful discussions; C. Chang, J. Doench, G. Barton, J. Jensen, J. Hurley, N. Mizushima, M. Ranaghan and F. Yarovinsky for providing critical reagents; L. Nguyen for technical assistance; and L. Bennett and H. Smith for assistance with manuscript preparation. This work was supported by NIH grants RO1 CA109618 (B.L.), U19 AI109725 (B.L), and U19 AI142784 (B.L.); Cancer Prevention Research Institute of Texas grant RP120718 (B.L.); and a Fondation Leducq grant 15CBD04 (B.L). M.J. was supported by a ‘chargé de recherché’ post-doctoral grant from the Belgian F.N.R.S.

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Contributions

Y.L., S.S.-K., M.H.R., S.J.M., P.M. and B.L. designed the study. Y.L., P.T.N., X.W., Y.Z., C.E.M., Z.Z., B.B., M.J., D.V., T.W., H.M. and S.S.-K. performed the experiments. Y.L., G.X. and B.L. analysed the data. Y.L. and B.L. wrote the manuscript.

Corresponding authors

Correspondence to Yang Liu or Beth Levine.

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Competing interests

B.L. is a Scientific Co-Founder of Casma Therapeutics, Inc.

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Extended data figures and tables

Extended Data Fig. 1 TLR7 and TLR9 interaction with beclin 1 and generation of TLR9–HA mice.

a, b, Co-immunoprecipitation of Flag–beclin 1 with TLR7–HA (a) or TLR9–HA (b) in transfected HeLa cells. c, Co-immunoprecipitation of TLR9–HA with Flag–full-length (FL) beclin 1 or deletion-mutant proteins in transfected U2OS cells. Flag–beclin 1(Δ244–266) is a control deletion mutant. d, Co-immunoprecipitation of TLR9–HA full-length protein or truncation mutant that lacks the TIR domain (1–868(ΔTIR)) with Flag–beclin 1 in transfected U2OS cells. e, MBP pull-down of recombinant beclin 1 with a protein comprising MBP fused to the TIR domain of TLR9 (MBP–TIR). f, Co-immunoprecipitation of indicated TLR9–HA constructs with indicated Flag–beclin 1 constructs in transfected U2OS cells cultured in normal medium, or subjected to 1-h glucose (−glu) or amino acid (−AA) starvation. g, Co-immunoprecipitation of TLR9–HA with Flag–beclin 1 in transfected U2OS cells cultured in normal medium, subjected to glucose starvation (for 1 h) or treatment for 1 h with the mitochondrial damaging agents oligomycin (2.5 μM) + antimycin A (250 nM) (OA) or CCCP (5 μM), or the direct AMPK activator PF-739 (5 μM). In ag, results are representative of three independent experiments. h, Tlr9 mRNA levels in different tissues. The value in lung tissue is considered to be 1. i, Tlr9 mRNA levels in myoblasts and myotubes (before and after myocyte differentiation, respectively) normalized to levels of β-actin. The value in myotubes is considered to be 1. In h, i, data are mean ± s.e.m. of triplicate samples. j, Schematic of the Tlr9 locus and CRISPR-based gene knock-in strategy (Methods). k, Representative genotyping of wild-type (+/+), heterozygous knock-in (+/KI) and homozygous knock-in (KI/KI) mice. l, Western blots of TLR9–HA expression in indicated tissues from mice (loading amount for spleen and lung, 20 μg; for heart and muscle, 50 μg). Full-length (approximately 130 kDa) and cleaved (approximately 80 kDa) TLR9 detected in spleen. In the skeletal muscle and heart tissues, only the predominant cleaved form of TLR9 was detected, owing to lower levels of TLR9 expression. Asterisk denotes a nonspecific band. m, Western blot indicating that full-length and cleaved TLR9 are detected in muscle lysates after enriching by immunoprecipitation with anti-HA antibody. For km, similar results were observed in three independent experiments. For uncropped gels, see Supplementary Fig. 1. Source data

Extended Data Fig. 2 TLR9–beclin 1 interaction and AMPK phosphorylation increases in skeletal muscle, but not in spleen, during exercise.

a, Representative western blots of endogenous beclin 1 co-immunoprecipitated with endogenous TLR9–HA in vastus lateralis muscles from TLR9–HA mice and wild-type mice (negative control) at indicated durations of exercise. Asterisk denotes a nonspecific band also observed in wild-type mice. Similar results were observed in three independent experiments. b, Quantification of p-AMPKα(T172)/total AMPKα (representative blots are shown in Fig. 1b) in vastus lateralis muscles from TLR9–HA mice at indicated durations of exercise. Results are combined data from three independent experiments with similar results in each experiment. c, d, Representative blots (c) and quantification (d) of p-AMPKα(T172)/total AMPKα in the spleen of TLR9–HA mice at indicated durations of exercise. Results are combined data from two independent experiments with similar results in each experiment. e, f, Representative western blots (e) and quantification (f) of beclin 1 co-immunoprecipitated with TLR9–HA in spleens from TLR9–HA mice at indicated durations of exercise. Results are combined data from two independent experiments with similar results in each experiment. In b, d, f, data points are individual mice (sample size is indicated in parentheses). Data are mean ± s.e.m. Values at 0 min are considered to be 1. In b, d, f, unpaired two-tailed t-test with Hommel method. For uncropped gels, see Supplementary Fig. 1. Source data

Extended Data Fig. 3 Levels of genomic DNA associated with TLR9 in skeletal muscle, levels of plasma mtDNA, and effects of treatment with exogenous TLR9 ligand on AMPK phosphorylation in skeletal muscle.

a, qPCR quantification of genomic DNA bound to TLR9. Cycle threshold (Ct) values for genomic DNA that co-immunoprecipitates with TLR9–HA from gastrocnemius muscles of wild-type or TLR9–HA mice at rest or after 20 min exercise are shown. Two sets of genomic DNA primers (Methods) were used. b, Quantification of mtDNA in plasma at serial time points after exercise  in wild-type mice. The amount of mtDNA was quantified by qPCR using six sets of mtDNA primers (Methods). For each primer set, the value at the 0-min time point is considered to be 1. In a, b, data are mean ± s.e.m.; data points are individual mice (n = 4). c, d, Representative blots (c) and quantification (d) of p-AMPK(T172)/total AMPK in EDL muscles from wild-type mice incubated ex vivo with 1 μM control ODN or ODN2395 (a TLR9 ligand) for 1 h. Data are mean ± s.e.m. Data points are individual muscles (n = 3). Unpaired two-tailed t-test. Western blots are from one representative experiment, and quantification data are combined from three independent experiments. Similar results were observed in each experiment. e, Western blot of AMPK phosphorylation at Thr172 in EDL muscles from wild-type mice incubated with or without 1 μM PF-739 (a direct AMPK activator) for 1 h. PF-739 is a positive control for experiments in c and d with respect to AMPK activation in EDL muscles ex vivo. Similar results were observed in three independent experiments. For uncropped gels, see Supplementary Fig. 1. Source data

Extended Data Fig. 4 Wild-type and Tlr9−/− mice display similar muscle characteristics and cardiac function.

a, b, Representative H&E staining (a) and fibre-type staining (b) of tibialis anterior muscles. In b, green denotes a plasma membrane marker (laminin); pink, type-I fibres (MHC I-positive); red, type-IIA fibres (MHC IIA-positive); blue, type-IIB fibres (MHC IIB-positive); and black, type-IIX fibres. c, Relative quantification of fibre-type composition in tibialis anterior muscles shown in b. Data are mean ± s.e.m. At least 1,400 muscle fibres were analysed per mouse. d, e, Representative images (d) and quantification (e) of staining for COX enzymatic activity (a measure of mitochondrial respiratory capacity) in tibialis anterior muscles. Data are mean ± s.e.m. At least 480 muscle fibres were analysed per mouse. f, g, Representative images (f) and quantification (g) of capillary density in tibialis anterior muscles. Data are mean ± s.e.m. At least 240 muscle fibres were analysed per mouse. h, Cardiac function determined by echocardiographic measurement of ejection fraction. For a, b, d, f, images are representative from one of two independent experiments. Similar results were observed in each experiment. Data are mean ± s.e.m. For c, g, h, data points are individual mice (n = 5 mice per group for c, h; n = 4 mice per group for g).  For  e, data points are the average values of five mice. Unpaired two-tailed t-test. Scale bars, 50 μm. Source data

Extended Data Fig. 5 Measurement of exercise-induced activation of AMPK in muscles, exercise-induced localization of GLUT4 to the plasma membrane in muscles, and maximal running distance in wild-type and Tlr9−/− mice.

a, b, Representative western blots (a) and quantification (b) of the phosphorylation of AMPK substrates (ACC at Ser79 and raptor at Ser792) in vastus lateralis muscles. The samples in a, b are the same as those used in Fig. 2a, b. c, d, Representative western blots (c) and quantification (d) of p-AMPK and p-TBC1D1 in EDL muscles. e, f, Western blots (e) and quantification (f) of GLUT4 levels in EDL muscles. Asterisk denotes a nonspecific band. g, Representative images of GLUT4 (red) and laminin (green) immunofluorescent staining (used for quantification in h and Fig. 2d) in EDL muscles. White denotes colocalization between GLUT4 and laminin as determined using ImageJ software. Scale bars, 20 μm. h, Percentage of total GLUT4 staining colocalized with laminin calculated using ImageJ software. At least 100 muscle fibres were analysed per mouse. i, Maximal running distance. Data are combined from three independent experiments. Similar results were observed in each experiment. For b, d, f, h, i, data are mean ± s.e.m.; data points are individual mice (sample size indicated in parentheses). In ad, g, h, western blots and images are from one representative experiment, and quantifications are combined data from three independent experiments. Similar results were observed in each experiment. In b, d, h, a two-tailed t-test was used to compare different conditions for each genotype and two-way ANOVA was used to compare magnitude of changes between different conditions in mice of different genotypes. In f, i, an unpaired two-tailed t-test was used. For uncropped gels, see Supplementary Fig. 1. Source data

Extended Data Fig. 6 Similar levels of adenine nucleotides, glycogen, total LKB1 and LKB1 phosphorylation at Ser428, and a similar response to an AMPK allosteric activator in wild-type and Tlr9−/− muscles.

ae, Measurements of ATP (a), ADP (b), AMP (c), ADP/ATP ratio (d) and AMP/ATP ratio (e) in EDL muscles. f, Glycogen content in tibialis anterior muscles. g, h, Representative western blots (g) and quantification (h) of p-LKB1(S428) and total LKB1 in vastus lateralis muscles. Representative blots are from one experiment, and quantification data are combined from three independent experiments. Similar results were observed in each experiment. i, j, Representative western blots (i) and quantification (j) of AMPK activation markers (p-AMPK and p-TBC1D1) in tibialis anterior muscles of mice, 90 min after subcutaneous administration of PF-739 (100 mg per 5 ml, per kg body weight). k, Blood glucose levels 90 min after treatment with PF-739. In ik, western blots are from one representative experiment, and quantification results are combined data from two independent experiments. Similar results were observed in each experiment. Data points are individual mice (sample size indicated in parentheses). Data are mean ± s.e.m. Unpaired two-tailed t-test. For uncropped gels, see Supplementary Fig. 1. Source data

Extended Data Fig. 7 Haematopoietic cells are not responsible for the defect in exercise-induced activation of AMPK in Tlr9−/− muscles, and TLR9 is required for ex vivo electrical-stimulation-induced activation of AMPK.

a, b, Percentage of wild-type Tlr9 genome (a) or Tlr9−/− genome (b) in blood cells from mice with indicated donor or recipient genotypes at eight weeks after bone marrow transplantation, as determined by qPCR. The copy number of Tlr7 genome is used as an internal control. Data are mean ± s.e.m. for five randomly selected mice per group. Data points are individual mice. The mean of wild-type–wild-type (a) or Tlr9−/−Tlr9−/− (b) donor–recipient combinations was considered to be 100%. c, d, Representative western blots (c) and quantification (d) of phosphorylation of TBC1D1 at Ser237 in vastus lateralis muscles of indicated recipient mice transplanted with indicated bone marrow cells, at rest and after 90 min exercise. e, f, Representative western blots (e) and quantification (f) of phosphorylation of TBC1D1 at Ser237 in EDL muscles of wild-type and Tlr9−/− mice with or without 20 min of electrical stimulation. In cf, western blots are from one representative experiment, and quantification results are combined data from three independent experiments. Similar results were observed for each experiment. Data points are individual mice (d) or muscles (f) (sample size indicated in parentheses). Data are mean ± s.e.m. In d, an unpaired two-tailed t-test was used to compare differences between donor genotypes, and a two-way ANOVA was used to compare differences between recipient genotypes. In f, an unpaired two-tailed t-test was used to compare conditions with and without electrical stimulation, for each genotype. A two-way ANOVA was used to compare the magnitude of changes between conditions with and without electrical stimulation in muscles of different genotypes. For uncropped gels, see Supplementary Fig. 1. Source data

Extended Data Fig. 8 Tlr9−/− mice have normal exercise-induced autophagy, and BCL2 binding to beclin 1 inhibits the beclin 1–TLR9 interaction induced by exercise or glucose starvation.

a, b, Representative images (a) and quantification (b) of GFP–LC3 puncta in vastus lateralis muscles of wild-type GFP-LC3 and Tlr9−/−;GFP-LC3 mice at rest or after exercise, with and without chloroquine pretreatment. Scale bars, 20 μm. Arrows mark representative puncta, counted in b. More than 15 randomly chosen fields were used per mouse and an average value was determined for each mouse. Images are from one representative experiment, and quantitative data are the combined results of two independent experiments. Similar results were observed in each experiment. Data points are individual mice (sample size in parentheses). c, d, Representative western blots (c) and quantification (d) of beclin 1 co-immunoprecipitated with TLR9–HA at serial time points after exercise in vastus lateralis muscles of TLR9–HA mice crossed to either BCL2 AAA mice or wild-type littermates. Western blots are from one representative experiment, and quantification data are the combined results from three independent experiments. Similar results were observed in each experiment. Data points are individual mice (sample size in parentheses). e, Co-immunoprecipitation of transiently expressed Flag–beclin 1 with Myc–BCL2 in HeLa cells stably expressing wild-type Myc–BCL2 or BCL2 AAA19 grown in normal medium or subjected to 2-h glucose starvation. Similar results were observed in three independent experiments. f, g, Representative western blots (f) and quantification (g) of Flag–beclin 1 co-immunoprecipitated with TLR9–HA in HeLa cells stably expressing wild-type Myc–BCL2 or BCL2 AAA grown in normal medium or subjected to 2-h glucose starvation. Data are mean ± s.e.m. from three experiments; sample size is indicated in parentheses. In b, an unpaired two-tailed t-test was used to compare different conditions for each genotype. A two-way ANOVA was used to compare the magnitude of changes between different conditions in mice of different genotypes. In d, a one-way ANOVA was used to compare 0-min and 20-min exercise conditions for each genotype. A two-way ANOVA was used to compare the magnitude of changes between 0- min and 20-min exercise conditions in mice of different genotypes. In g, an unpaired two-tailed t-test was used. For uncropped gels, see Supplementary Fig. 1. Source data

Extended Data Fig. 9 The beclin 1 and UVRAG interaction increases during exercise and during muscle contractions induced by electrical stimulation.

a, b, Representative blots (a) and quantification (b) of UVRAG co-immunoprecipitated with beclin 1 in tibialis anterior muscles of wild-type mice at indicated durations of exercise. Western blots are from one representative experiment, and quantification data are the combined results from five independent experiments. Similar results were observed in each experiment. c, d, Representative blots (c) and quantification (d) of beclin 1 co-immunoprecipitated with UVRAG in EDL muscles of wild-type and Tlr9−/− mice with or without 20 min of electrical stimulation. Western blots are from one representative experiment, and quantification data are the combined results from two independent experiments. Similar results were observed in each experiment. In b, d, data points are individual mice (b) or muscle (d) (sample size indicated in parentheses). Data are mean ± s.e.m. In b, an unpaired two-tailed t-test was used. In d, a two-tailed t-test was used to compare conditions with and without electrical stimulation for each genotype. A two-way ANOVA was used to compare the magnitude of changes in conditions with and without electrical stimulation in muscles of different genotypes. Asterisk denotes a nonspecific band observed in IgG control condition. For uncropped gels, see Supplementary Fig. 1. Source data

Extended Data Fig. 10 Beclin 1, UVRAG or ATG14 knockout does not affect levels of LKB1 protein or the phosphorylation of LKB1 at Ser428 induced by glucose starvation.

ac, Effects of beclin 1 (a), UVRAG (b) or ATG14 (c) knockout in U2OS cells on levels of LKB1 protein and phosphorylation of LKB1 at Ser428, 1 h after glucose starvation. Left, representative western blots of p-LKB1, total LKB1 and total AMPKβ in cells with indicated gene knockout (two independent gRNAs per target gene). Right, quantification of total LKB1/total AMPKβ (from four independent experiments) and p-LKB1/total LKB1 (from three independent experiments). Similar results were observed in each experiment. Data are mean ± s.e.m. Sample size is indicated in parentheses. Unpaired two-tailed t-test. NG, no gRNA. For uncropped gels, see Supplementary Fig. 1. Source data

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Liu, Y., Nguyen, P.T., Wang, X. et al. TLR9 and beclin 1 crosstalk regulates muscle AMPK activation in exercise. Nature 578, 605–609 (2020). https://doi.org/10.1038/s41586-020-1992-7

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