The phosphorylation of AMPKβ1 is critical for increasing autophagy and maintaining mitochondrial homeostasis in response to fatty acids

Abstract

Fatty acids are vital for the survival of eukaryotes, but when present in excess can have deleterious consequences. The AMP-activated protein kinase (AMPK) is an important regulator of multiple branches of metabolism. Studies in purified enzyme preparations and cultured cells have shown that AMPK is allosterically activated by small molecules as well as fatty acyl-CoAs through a mechanism involving Ser108 within the regulatory AMPK β1 isoform. However, the in vivo physiological significance of this residue has not been evaluated. In the current study, we generated mice with a targeted germline knock-in (KI) mutation of AMPKβ1 Ser108 to Ala (S108A-KI), which renders the site phospho-deficient. S108A-KI mice had reduced AMPK activity (50 to 75%) in the liver but not in the skeletal muscle. On a chow diet, S108A-KI mice had impairments in exogenous lipid-induced fatty acid oxidation. Studies in mice fed a high-fat diet found that S108A-KI mice had a tendency for greater glucose intolerance and elevated liver triglycerides. Consistent with increased liver triglycerides, livers of S108A-KI mice had reductions in mitochondrial content and respiration that were accompanied by enlarged mitochondria, suggestive of impairments in mitophagy. Subsequent studies in primary hepatocytes found that S108A-KI mice had reductions in palmitate- stimulated Cpt1a and Ppargc1a mRNA, ULK1 phosphorylation and autophagic/mitophagic flux. These data demonstrate an important physiological role of AMPKβ1 Ser108 phosphorylation in promoting fatty acid oxidation, mitochondrial biogenesis and autophagy under conditions of high lipid availability. As both ketogenic diets and intermittent fasting increase circulating free fatty acid levels, AMPK activity, mitochondrial biogenesis, and mitophagy, these data suggest a potential unifying mechanism which may be important in mediating these effects.

Keywords: AMPK; NAFLD; autophagy; fat oxidation; mitochondria.

Fig. 1.

AMPKβ1 Ser108 phosphorylation is important for acute fatty acid-induced increases in ACC phosphorylation and whole-body fatty acid oxidation. (A) AMPKα isoform-specific phosphotransferase activity assay from basal chow-fed (CD) WT and S108A-KI (KI) mice for liver and quadriceps muscle (Quad). (B–D) Schematic of fast-refeed experiments with or without intralipid or AICAR in metabolic monitoring units (B). Fatty acid oxidation (C) was calculated from VO₂ and VCO₂ over 4 h, starting 1 h postgavage of either saline or 20% intralipid (10 mL/kg) in WT and S108A-KI mice fed a chow diet. Following an i.p. injection of saline or AICAR (500 mg/kg), fatty acid oxidation (D) was calculated from VO₂ and VCO₂ over 1 h, starting 6 h postinjection. Representative ACC immunoblotting and densitometrical analysis assessing inhibitory phosphorylation of ACC by AMPK in response to palmitate (Palm: 500 µM) (E), AICAR (AIC: 100 µM) (F), and A-769662 (A76: 10 µM) (G) in primary hepatocytes from CD-fed WT and S108A-KI mice. Data are means ± SEM with P-values reported in the graphs. Gray bars equal WT differences, blue bars equal to S108A-KI differences, and black bars indicate differences between groups in same treatment condition. Statistical significance was accepted at P < 0.05 and determined via multiple t tests or two-way ANOVA with Tukey’s posthoc analysis. White circles are individual mice or experimental replicates with three technical replicates per group.

Fig. 2.

AMPKβ1 Ser108 phosphorylation is important for increasing mitochondrial biogenesis in mice fed a HFD or hepatocytes treated with palmitate. (A) Representative immunoblots of ACC1/2 S79/S212 and AMPKβ1 S108 and densitometrical analysis of pACC/ACC (B) and total ACC (C) from the livers of chow-fed (CD) and HFD-fed (HFD) WT and S108A-KI mice. (D) Intraperitoneal glucose tolerance test (Intraperitoneal glucose tolerance tests (ipGTT), 0.8 g/kg) and area under the curve (GTT AUC) at 24 wk of age in HFD-fed WT and S108A-KI mice. (E) Serum insulin at 2- and 10-min post glucose injection from some of the mice in D in which blood samples could be collected. Representative H&E stains (10×, F) and liver triglycerides (G) of WT and S108A-KI mice fed a HFD for 20 wk. Representative immunoblots and densitometrical analysis of OXPHOS complexes 2-5 of WT and S108A-KI mice fed a HFD (H). mRNA expression of peroxisomeproliferator activated receptor gamma coactivator 1-alpha (Ppargc1a) in WT and S108A-KI mice fed a HFD (I). (J–L) Schematic representation of mRNA experiments in primary hepatocytes for mitochondrial biogenesis in response to elevated LCFAs (J). Carnitine-palmitoyl transferase 1-alpha (Cpt1a) (K) and Ppargc1a (L) mRNA expression from primary hepatocytes. Data are means ± SEM with P-values reported in the graphs. Gray bars equal WT differences, blue bars equal to S108A-KI differences, and black bars indicate differences between groups in same treatment condition. Significance was accepted at P < 0.05 and determined via t tests, ordinary two-way, or repeated-measures two-way ANOVA with Tukey’s posthoc analysis, where appropriate. White circles are individual mice per group. Black arrow in A indicates area for specific band of AMPKβ1 S108.

Fig. 3.

AMPKβ1 Ser108 phosphorylation is important for increasing ULK1 phosphorylation and maintaining mitochondrial morphology and function in response to a HFD. Respiration rates (A) from isolated liver mitochondria from CD and HFD-fed WT and S108A-KI mice (PM; 0.5 mM malate, 5 mM pyruvate, PMD; + 1 mM ADP, +Glu; 5 mM glutamate, +Oligo; 1.25 µM oligomycin, +FCCP; titration of 0.5 µM FCCP until maximal uncoupled respiration reached, +Succ; 10 mM succinate, and +Mal; 5 mM malonate). Representative electron micrographs (B) with quantification of average mitochondrial surface area (C), mitochondrial number (D), and mitochondrial surface area per field (E) from WT and S108A-KI mice fed a HFD. Representative immunoblotting (F) and densitometrical analysis of pULK S555/ULK1 (G) and LC3BII (H) from the livers of WT and S108A-KI mice fed a HFD. Data are means ± SEM with P-values reported in the graphs. Gray bars equal WT differences and blue bars equal to S108A-KI differences. Statistical significance was accepted at P < 0.05 and determined via unpaired t test, two-way ANOVA with Tukey’s posthoc analysis, or repeated-measures two-way ANOVA with Sidak’s posthoc analysis, where appropriate. White circles are individual mice or technical replicates from three experimental replicates per group.

Fig. 4.

AMPKβ1 Ser108 phosphorylation is important for increasing autophagy and mitophagy in response to increases in fatty acid availability. (A–F) Schematic of immunoblotting experiments for palmitate-induced autophagy and autophagic flux experiments in primary hepatocytes (A). Representative immunoblots (B) and densitometrical analysis of ULK1 (S555) (C) and ATG14 (S29) (D) assessing the induction of autophagy via AMPK activation in response to palmitate (500 µM) in primary hepatocytes from CD-fed WT and S108A-KI mice. Representative LC3B-II immunoblotting and densitometrical analysis (E) assessing autophagic flux in response to palmitate (500 µM) in primary hepatocytes from CD-fed WT and S108A-KI mice. (F–H) Schematic of palmitate-induced autophagic flux in primary hepatocyte experiments by tandem fluorescence and graphical illustrating result of using the LC3B protein with an acid-sensitive GFP and an acid-insensitive RFP (F). Representative colorblindness-friendly confocal micrographs with green being replaced with cyan (autophagosomes appear as white, while autophagolysosomes are red) (G), and quantification of percent red not overlapping green (H) in CD-fed WT and S108A-KI primary hepatocytes treated with vehicle or palmitate. (I–K) Schematic of immunoblotting experiments for palmitate-induced mitophagic flux experiments in primary hepatocytes (I). Representative immunoblotting (J) and densitometrical analysis of LC3B-II (K) assessing mitophagic flux in response to palmitate (500 µM) in primary hepatocytes from CD-fed WT and S108A-KI mice. Working model of the role of AMPKβ1 Ser108 phosphorylation in an AMPK-fatty acid-sensing axis to increase fatty acid oxidation, autophagy/mitophagy, and mitochondrial biogenesis (L). Data are means ± SEM with P-values reported in the graphs. Gray bars equal WT differences, blue bars equal to S108A-KI differences, and black bars indicate differences between groups in same treatment condition. A straight black line indicates a main effect of genotype in D. Statistical significance was accepted at P < 0.05 and determined via repeated-measures two-way ANOVA with Sidak’s posthoc analysis, unpaired t tests, or two-way ANOVA with Tukey’s posthoc analysis, where appropriate. White circles are individual mice or technical replicates from experiments with three experimental replicates per group. For confocal microscopy analysis, white circles represent individual micrographs from two experimental replicates per group.