Fig 2: Independently of AMPK, metformin activates protein phosphatase 2A (PP2A) in inguinal white adipose tissue (iWAT). (A-D) Representative western blots of phosphorylated AMP-activated protein kinase (Thr 172; p-AMPK/t-AMPK), mammalian target of rapamycin complex 1 (mTORC1), fatty acid synthase (FASN), phosphorylated acetyl-CoA carboxylase (Ser 79; p-ACC/t-ACC) and phosphorylated hormone sensitive lipase (Ser 660; p-HSL/t-HSL) in iWAT. (E) Ten mg of iWAT was homogenized and PP2A immunoprecipitated for activity measurements using malachite green. Densitometric measurements of western blots were performed using ImageJ. Image 2: control (n = 8); Image 5: control + metformin (n = 8) Image 3: burn (n = 8); Image 4: burn + metformin treatment (n = 8). Values are presented as mean ± standard error. *p ≤ 0.05; **p ≤ 0.01.

Fig 3: Skeletal muscle–specific overexpression of ATGL and its impact on lipid homeostasis. A: The Ckm-ATGL transgene construct. B: ATGL mRNA expression relative to 18S control gene by quantitative PCR in muscle and nonmuscle tissues with endogenous ATGL expression in quadriceps arbitrarily set to 1 (Line 1, ♀, 8 weeks, chow, fasted 12 h; n = 4–5/group). The fold-increase in ATGL mRNA expression in Tg relative to WT mice for select muscle tissues in each of two founder lines is shown in the table (bottom). C: ATGL protein expression relative to Ran GTPase (RAN) control as determined by immunoblotting in skeletal versus cardiac muscle (Line 1, ♀, 17 weeks, chow, fasted 12 h, gastrocnemius and heart; n = 6/group). D: ATGL IF in skeletal muscle (Line 1, ♂, 17 weeks, chow, fasted 12 h, gastrocnemius). E: Quantitative fiber type–specific ATGL protein expression by IF (Line 1, ♂, 28 weeks, chow and HFD, fasted 12 h, gastrocnemius-plantaris-soleus [GPS] complex, average of four muscle areas each; n = 4/group). Type 2B fibers of chow-fed WT mice are arbitrarily set to 1. F: TAG hydrolase activity in the absence (basal) and presence of the HSL-specific inhibitor 76-0079 (Line 1, ♂, 12 weeks, chow, fasted 12 h, gastrocnemius; n = 3/group). G: Skeletal muscle histology of control (left) and Tg (right) mice including general morphology by H&E staining (top) and IMTG content by ORO staining (bottom) (Line 1, ♂, 12 weeks, HFD for 4 weeks, fasted 12 h, GPS complex). H: IMTG content by ORO staining using quantitative IF (Line 1, ♂, 28 weeks, chow and HFD, fasted 12 h, GPS complex, average of four muscle areas each; n = 4/group). Type 2B fibers of chow-fed WT mice are arbitrarily set to 1. I: Intramyocellular DAG, ceramide, and FA-CoA content using biochemical analysis of whole muscle (Line 1, ♂, 28 weeks, chow and HFD, fasted 12 h, quadriceps; n = 3/group). For overall effects having P < 0.05: D, diet; F, fiber type; G, genotype; T, treatment (with HSL-inhibitor). For specific comparisons having P < 0.05: #for effect of diet; *for effect of genotype; and @for effect of treatment. AU, arbitrary units; BAT, brown adipose tissue; EDL, extensor digitorum longus; Gas, gastrocnemius; Hrt, heart; Panc, pancreas; PGAT, perigonadal adipose tissue; Quad, quadriceps; Sol, soleus; TA, tibialis anterior.

Fig 4: Skeletal muscle mitochondrial function and expression of genes/proteins regulating lipid homeostasis in SMAKO mice. A: FA oxidation (♂, 28 weeks, fasted 12 h, red gastrocnemius; n = 6/group). 14C-labeled incorporation into CO2 and acid-soluble metabolites (ASMs) represent complete and incomplete oxidation, respectively. B: Mitochondrial respiration in permeabilized muscle fibers (♂, 28 weeks, fasted 12 h, soleus; n = 6/group). Oxygen consumption was measured following the sequential addition of the following substrates: palmitoylcarnitine (P), malate (M), ADP (D), glutamate (G), succinate (S), and cytochrome c, oligomycin, and carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP). The corresponding respiratory states are noted: ADP-driven respiration (state 3), respiration in the absence of ADP (state 4), and uncoupled respiration (state U). C: Expression of oxidative phosphorylation proteins in complexes I–V (NDUFB8 [complex I], SDHB [complex II], UQCRC2 [complex III], MTCO1 [complex IV], and ATP5A [complex V]) (♂, 28 weeks, fasted 12 h, tibialis anterior; n = 6/group). Data are normalized to protein expression of β-actin. D: TEM of skeletal muscle (♂, 28 weeks, fasted 12 h, red quadriceps, representative images). mRNA expression of PPARα/PGC1α and their target genes (E) and genes for lipid breakdown (lipolysis), uptake, and synthesis as well as lipid droplet–associated proteins (LDAPs) of the Plin family (F) relative to cyclophilin control with expression in WT-chow normalized to 1 (♂, 28 weeks, tibialis anterior; n = 9–13/group). G: Protein expression of total HSL normalized to Ran GTPase (RAN) control (left), phosphorylated HSL normalized to total HSL (middle), and representative immunoblots (right) (♂, 28 weeks, tibialis anterior; n = 4/group). For mRNA and protein expression, samples were confirmed to have low or no expression of Plin1, thereby confirming absence of significant fat contamination. For overall effects having P < 0.05: D, diet; G, genotype. Where an interaction was identified, specific comparisons having P < 0.05: *for effect of genotype.

Fig 5: Skeletal muscle mitochondrial function and expression of genes/proteins regulating lipid homeostasis in Ckm-ATGL mice. A: FA oxidation (♂, 28 weeks, fasted 12 h, red gastrocnemius; n = 5–8/group). B: Mitochondrial respiration in permeabilized muscle fibers (♂, 34 weeks, fasted 12 h, extensor digitorum longus; n = 3–6/group). C: Expression of oxidative phosphorylation proteins in complexes I–V (♂, 34 weeks, fasted 12 h, tibialis anterior; n = 4–7/group). Data are normalized to protein expression of β-actin. D: TEM of skeletal muscle (♂, 34 weeks, fasted 12 h, red quadriceps, representative images). mRNA expression of PPARα/PGC1α and their target genes (E) and genes for lipid breakdown (lipolysis), uptake, and synthesis as well as lipid droplet–associated proteins (LDAPs) of the Plin family (F) relative to cyclophilin control with expression in WT-chow normalized to 1 (♂, 34 weeks, tibialis anterior; n = 7–21/group). G: Protein expression of total HSL normalized to Ran GTPase (RAN) control (left), phosphorylated HSL normalized to total HSL (middle), and representative immunoblots (right) (♂, 34 weeks, tibialis anterior; n = 5–7/group). For mRNA and protein expression, samples were confirmed to have low or no expression of Plin1, thereby confirming absence of significant fat contamination. For overall effects having P < 0.05: D, diet; G, genotype.