Eric M. Desjardins, Brennan K. Smith, Emily A. Day, Serge Ducommun, Matthew J. Sanders, Joshua P. Nederveen, Rebecca J. Ford, Stephen L. Pinkosky, Logan K. Townsend, Robert M. Gutgesell, Rachel Lu, Kei Sakamoto, and Gregory R. Steinberg

November 21, 2022; 119 (48) e2119824119. https://doi.org/10.1073/pnas.2119824119

Significance

The data in this manuscript indicate a crucial role for a single phosphorylation site on the regulatory beta subunit of AMPK to stimulate mitochondrial biogenesis and autophagy/mitophagy in response to increases in fatty acids. This suggests a potential unifying mechanism which may be important for mediating the beneficial effects of dietary interventions that increase free-fatty acid availability such as intermittent fasting and ketogenic diets.

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.

See https://www.pnas.org/doi/10.1073/pnas.2119824119

Figure 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.