Fig 2: Regulation of lipid and glucose metabolism by MKRN1 and AMPK. a, b Relative proportions of DEGs in MKRN1-null liver and adipose tissues according to their associated GOBPs. The GOBP terms at level 1 (a) and levels 2–4 (b) were used for general cellular processes and metabolic processes, respectively. c Cellular processes related to lipid and carbohydrate metabolism enriched by the up- and downregulated genes identified in MKRN1-null liver or adipose tissue. The bars represent −log10 (P-value), where the P-value is the significance of the processes being enriched by the up- or downregulated genes. d Heat maps showing the changes in the expression of DEGs involved in fatty acid biosynthesis and β-oxidation, gluconeogenesis and thermogenesis. The colour bar shows the gradient of the log2 fold changes of mRNA expression levels in MKRN1-depleted samples relative to those in WT samples. e, f Network models describing alterations of metabolic reactions regulated by DEGs in MKRN1-null livers (e) and adipose tissue (f). Arrows denote metabolic reactions, and dotted lines denote the transportation of molecules or regulation involving intermediate regulators between the linked molecules. Node colours represent up- (red) or downregulation (green) in MKRN1-null livers or adipose tissue. The colour bar represents the gradient of the log2 fold changes of mRNA expression levels induced by MKRN1 ablation relative to those in WT

Fig 3: Down-regulation of Angptl4 expression by AMPK is likely mediated via inhibition of PPARγ-mediated transcription of Angptl4.(A) Angptl4 mRNA in differentiated T37i adipocytes pre-incubated with 0.5 μg/mL actinomycin D (ActD) or DMSO control for 1 hr and treated with 1 mM AICAR or control for 3 hr. (B) Angptl4 mRNA in differentiated T37i or BA adipocytes treated with DMSO control, 5 μM rosiglitazone, 10 μM Wy14643, or 5 μM GW742 (BA adipocytes) or 5 μM GW501516 (T37i adipocytes) for 6 hr. (C) Immunoblot for ANGPTL4 in differentiated T37i adipocytes treated with 5 μM rosiglitazone (Rosi), 10 μM Wy14643 (Wy), and 5 μM GW501516 for 24 hr. (D) Angptl4 mRNA in differentiated T37i cells treated with DMSO control, 5 μM rosiglitazone (Rosi), 1 mM AICAR or both rosiglitazone and AICAR for 6 hr. (E) Relative luciferase activity of HepG2 cells transfected with pGL3-Angptl4, pSG5-Pparγ, pSG5-Rxr, pcDNA-P300HA vectors, as indicated and treated 16 hr post-transfection with 5 μM rosiglitazone, 1 mM AICAR or both compounds for 9 hr. Data are represented as ± SD. (F) Immunoblot of P300 and phospho-P300 (Ser-89) in differentiated T37i brown adipocytes treated with control or 1 mM AICAR for 3 hr. *Statistically significant compared to control samples or between indicated samples according to Student’s t-test (p<0.05). Error bars represent ± SEM, unless otherwise specified.DOI: http://dx.doi.org/10.7554/eLife.08428.014

Fig 4: Identification of enymatically active AMPK on OMM in vivo. (A) Whole-cell lysates (WC), postnuclear lysates (PNL), and the corresponding enriched mitochondrial fractions of Percoll gradient isolation from mouse GA and Heart were probed for pan-AMPKα with Vdac, Catalase, and α-tubulin as loading and purity controls (n = 3). CS denotes mixed whole-tissue lysate comprised of mouse skeletal muscle, heart, and liver. (B) Immunofluorescence confocal microscopy of longitudinal sections of C57BL/6 mouse plantaris muscle probed by pan-AMPKα (red) and Cox4 (green) antibodies and DAPI for nuclear DNA (blue). Representative image of n = 3. (Scale bar, 20 µm.) (C) Enriched mitochondrial and cytosolic fractions isolated via differential centrifugation from mouse GA muscle and probed for each AMPK subunit isoform. n = 3. (D) Enriched mitochondrial fractions from frozen GA muscle of AMPKβ1/β2 knockout (KO) and wild-type littermate mice (WT). n = 3 per group. (E) Enriched mitochondrial fractions from mouse GA were treated with/without trypsin and probed for AMPKα1/α2/β2/γ1 (n = 2 per condition). An illustration of the physical association of AMPK with OMM is presented below. (F) Enriched mitochondrial fractions from human skeletal muscle biopsies (n = 2) and left ventricle biopsies (n = 3) were probed for AMPKα1/α2/β2/γ1. (G) AMPK activity in WC, cytosolic (Cyto), and enriched mitochondrial (Mito) factions with (+) and without (-) AMP (n = 3). All data presented as mean ± SEM. ***P < 0.001 by two-way ANOVA.

Fig 5: FKBP51 associates with amino acids and polyamine biosynthesis pathways.(A) Analysis and regulation of significantly altered pathways of FKBP51 KO and WT cells. The f(x) axis shows the (median) log2 fold change (FC) of all significantly altered metabolites of the indicated pathway, and the false discovery rate (FDR) (equals the −log10–adjusted P value) is shown on the x axis. The size of the circles represents the amount of significantly changed metabolites in comparison to all metabolites of a particular pathway. tRNA, transfer RNA. (B) FKBP51 deletion increases metabolites of the polyamine pathway, the AMP/ATP ratio, and enhances levels of amino acids associated with mTOR signaling. Data in (B) are shown as means + SEM and were analyzed by a two-way analysis of variance (ANOVA) and a subsequent Bonferroni multiple comparison analysis. ala, alanine; AMPK, AMP-activated protein kinase; arg, arginine; asp, asparagine; bio., biosynthesis; CoA, coenzyme A; cys, cysteine; dcSAM, decarboxylated S-adenosylmethione; gln, glutamine; glu, glutamic acid; gly, glycine; GSH, glutathione (reduced); his, histidine; ile, isoleucine; leu, leucine; LKB1, liver kinase B1; met, methionine; met., metabolism; MTA, 5′-methylthioadenosine; mTORC1, mechanistic target of rapamycin complex 1; NAput, N-acetylputrescine; NAspd, N-acetylspermidine; NAspm, N- acetylspermine; orn, ornithine pro, proline; phe, phenylalanine; put, putrescine; SAM, S- adenosylmethionine; ser, serine; spd, spermidine; spm, spermine; TFEB, transcription factor EB; TSC2, tuberous sclerosis complex 2; thr, threonine; trp, tryptophan; tyr, tyrosine; val, valine. *P < 0.05, **P < 0.01.