Journal: Signal Transduction and Targeted Therapy

Article Title: A two-strata energy flux system driven by a stress hormone prioritizes cardiac energetics

doi: 10.1038/s41392-025-02402-9

Figure Lengend Snippet: FGF21 deficiency alters systemic metabolites linked to mitochondrial and cardiac energetic defects. a – c Heatmap of significantly altered serum metabolites, including mitochondrial FAO precursors, fatty acids and oxylipins, TCA cycle intermediates, and nucleosides, in Fgf21 −/− (n = 3) and WT (n = 3) mice. p < 0.05; fold change (FC) > 1.5 or < 0.67. See also Fig. S a–c and S . For changes in serum phosphocreatine levels, see Fig. S . S, supplementary. d Experimental procedure for electrocardiogram analysis under anesthesia and anabiosis simulating ‘sleep’ (anesthesia on) and ‘awakening’ (anesthesia off) stages of torpor. ‘Sleep’, a sedentary state under anesthetic inhalation; Awakening, a recovery and active state following anesthetic removal. e Heart rate (HR) changes in Fgf21 −/− (n = 95) vs WT (n = 56) mice measured in d under normal conditions. BPM, beats per minute. f Area-under-curve (AUC) analysis of the HR excursion curves in e. See Fig. S . g , h Echocardiogram analysis of the mechano-energetic efficiency (MEE), ejection fraction (EF) and cardiac output (CO) of Fgf21 -/- (n = 51) vs WT (n = 51) mice. See Fig. S b-S for other Echo parameters. i Telemetry monitoring of HR changes over 24 hours in representative Fgf21 -/- vs WT mice under normal conditions. j AUC analysis of cumulative HR excursion curves of Fgf21 -/- (n = 6) vs WT (n = 6) mice in i. Changes in ambulatory movement and body temperature are shown in Fig. S j-S . k Representative TEM images of left ventricle cross-sections from Fgf21 -/- vs WT mice under normal conditions. Yellow arrowhead, mitochondria. Cyan arrowhead, Z line. See Fig. S for broader, low-magnification images. The changes in cardiac morphometric parameters, mild dilatation and insignificant fibrosis are shown in Fig. S . l Mitochondrial DNA content of hearts from Fgf21 -/- (n = 11-12) vs WT (n = 12) mice. m Catalytic activities of mitochondrial complexes I-IV in the hearts of Fgf21 -/- (n = 7-9) vs WT (n = 7-9) mice. h, hour. n , o HR Changes (n) with rhFGF21 treatment (n = 12) and AUC analysis (o) of Fgf21 -/- (n = 78) vs WT (n = 47) mice under normal conditions. p , q Changes in EF and LVSED with rhFGF21 treatment (n = 22) in Fgf21 -/- (n = 51) vs WT (n = 51) mice, as in n. See Fig. S g-S for other parameters. The data are presented as the means ± s.e.m.s. ( a – c , e – j ) Two-tailed unpaired Student’s t-test; ( l – q ) ordinary one-way ANOVA followed by Tukey’s test; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. *, Fgf21 -/- vs WT groups by Student’s t test or one-way ANOVA. $ , between treatment groups in WT mice. # , between treatment groups in Fgf21 -/- mice. All these also apply to other figures

Article Snippet: To establish a mouse model with mTOR inhibition, 8-week-old Fgf21 -/- mice were injected with Torin-1 (0.05 mg/mouse, i.p .) (MedChemExpress, USA) followed by a 12-h fast.

Techniques: Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: A two-strata energy flux system driven by a stress hormone prioritizes cardiac energetics

doi: 10.1038/s41392-025-02402-9

Figure Lengend Snippet: FGF21 orchestrates cardiac energy flux by engaging the LKB1-AMPK-mTOR energy stress pathways. a Transcriptomic enrichment of the LKB1-AMPK and mTOR energy regulation pathways in the hearts of fasted Fgf21 -/- (n = 3) vs WT (n = 3) mice and those with rhFGF21 treatment (n = 3). b Representative Western blot analysis of total kinase activities of cardiac LKB1, AMPK, and mTOR in the indicated mice (n = 3-6 for each group). Total kinase activity = (p-Kinase/Kinase) x (Kinase/Beta-actin), see also Fig. S . c Effects of cardiac-specific Stk11 (LKB1) ablation ( Stk11 f/f ;Myh6 Cre ) on HR. AUC analysis on the right. See Fig. S for experimental scheme, Fig. S for the AUC in the ‘Sleep’ and Awakening phases, and Fig. S29d for the Echo parameters. d Representative Western blot analysis of total kinase activities of cardiac AMPK, and mTOR in the indicated groups of mice (n = 3-6 for each group). See Fig. S . e Expression of representative genes involved in the TCA cycle, ETC, OXPHOS, and heart contraction in fasted Stk11 f/f ;Myh6 Cre and Fgf21 -/- mice 2 hours after rhFGF21 treatment. For changes in cardiac myofibrillar and mitochondrial structure, see Fig. S . Effects of mTOR inhibitor Torin-1 (0.05 mg per mouse, i.p .) on rhFGF21-induced HR improvements in fasted Fgf21 -/- mice (n = 11-12 per group). See Fig. S f, for experimental scheme and total AUC. Effects of Torin-1 and liver-specific Hmgcs2 deficiency on the cardiac ATP content in acute rhFGF21-treated Fgf21 -/- mice (n = 6 per group) following 12-h and 24-h fasts, respectively. h Effects of the AMPK activator AICAR (1.25 mg/mouse, i.p .) (n = 6) on rhFGF21-induced HR improvements in fasted Fgf21 -/- mice (n = 12 and 17, respectively). See Fig. S b-S for experimental scheme and total AUC, and Fig. S30d for the Echo parameters. i Transcriptomic enrichment of mitophagy, macroautophagy and associated LKB1-AMPK-mTOR energy stress pathways in the mice as in a. See Fig. S e, for mitophagy regulation by LKB1-AMPK-mTOR and autophagosome assembly. Data are means ± s.e.m.s; (d, e ) Two-tailed unpaired Student’s t-test; ( a – c , - i ) ordinary one-way ANOVA followed by Tukey’s test

Article Snippet: To establish a mouse model with mTOR inhibition, 8-week-old Fgf21 -/- mice were injected with Torin-1 (0.05 mg/mouse, i.p .) (MedChemExpress, USA) followed by a 12-h fast.

Techniques: Western Blot, Activity Assay, Expressing, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: A two-strata energy flux system driven by a stress hormone prioritizes cardiac energetics

doi: 10.1038/s41392-025-02402-9

Figure Lengend Snippet: FGF21 deficiency impairs cardiac energetic performance under various physiologically relevant stress conditions. a Experimental outline for assessing heart function following 12 hours (h) of fasting. Note that food deprivation for both 12 and 24 hours is referred to as fast (fs) hereafter, except where time constraints are specified (fast, 12-h; fast, 24-h; starvation, 48 h or 2 days). All experiments, except refeeding, were conducted while the mice were still in fasting/starvation after the initial food-deprivation period. b Heart rate (HR) changes during fasting in Fgf21 -/- (n = 17) vs WT (n = 17) mice. c Comparative HR changes in fasted Fgf21 -/- mice after refeeding and rhFGF21 treatment (1 mg/kg, i.p .). For HR changes after a 24-h fast, see Fig. S . d Area-under-curve (AUC) analysis of HR changes in fasted Fgf21 -/- vs WT mice after refeeding and rhFGF21 treatment for 2 hours. See Fig. S for more detailed HR comparisons and S4b for simplified AUC plots. , f Changes in ejection fraction (EF) and cardiac output (CO) under the same conditions as in c. Fgf21 -/- refeeding, n = 27; rhFGF21, n = 28; WT, rhFGF21, n = 38. See Fig. S c– . g HR changes measured by telemetry in response to involuntary running in Fgf21 -/- (n = 7) vs WT (n = 11) mice after 24 hours of fasting. For the experimental scheme see Fig. S . h Changes in running speed (steps per minute) in the indicated mice as in g . i Changes in running duration (total steps in 30 minutes) in the indicated mice as in g. For body temperature changes, see Fig. S . j Impacts of endurance training (2 hours/day for 56 days) on the HR (AUC) in Fgf21 -/- (n = 11) vs WT (n = 8) mice. For HR changes at 7, 14, 28 days, see Fig. S c– . k HR changes after 56 days of endurance training in the indicated mice. d, day. l , m Changes in EF and LVESD after 28 days of endurance training in the indicated mice. See Fig. S a– . n Changes in core body temperature in Fgf21 -/- (n = 7) vs WT (n = 6) mice subjected to a 4 °C challenge for 48 hours. ΔT, core body temperature change between 4 °C and room temperature, as measured by a rectum probe. See Fig. S . o HR changes measured by telemetry in Fgf21 -/- (n = 8) vs WT (n = 8) mice after 24 hours of fasting and then 2 hours of exposure to 4°C. See Fig. S g-S . p Changes in core body temperature in the indicated mice as in o. See Fig. S . q Changes in cumulative pedestrian activities (total steps in 2 hours) in the indicated mice as in o . See Fig. S . Data are means ± s.e.m.s; ( b , g – q ) Two-tailed unpaired Student’s t -test; ( c – f ) ordinary one-way ANOVA followed by Tukey’s test

Article Snippet: To establish a mouse model with mTOR inhibition, 8-week-old Fgf21 -/- mice were injected with Torin-1 (0.05 mg/mouse, i.p .) (MedChemExpress, USA) followed by a 12-h fast.

Techniques: Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: A two-strata energy flux system driven by a stress hormone prioritizes cardiac energetics

doi: 10.1038/s41392-025-02402-9

Figure Lengend Snippet: FGF21 restoration improves heart dysfunction under stress by promoting mitochondrial energy flux and macronutrient flexibility. a Effects of increased energy flux via a high-fat diet (HFD) and rhFGF21 on heart rate (HR) in Fgf21 -/- (n = 12) vs WT (n = 12) mice. w, week. See Fig. S . b Area-under-curve (AUC) analysis of HR changes in a. See Fig. S . c , Changes in EF and CO in the same groups as in a. See Fig. S . , f Changes in serum triglycerides (TG) and free fatty acids (FFA) in Fgf21 -/- (n = 6) vs WT (n = 6) mice as in a, as well as after 3-week rhFGF21 treatment. For other hepatic and serum metabolic parameters, see Fig. S a– . For the HFD effects on cardiac mitochondria, see Fig. S . For glucose intolerance status, see Fig. S i, . Time courses of the effects of FGF21 restoration on HR via AAV-mediated overexpression in Fgf21 -/- (n = 12) vs AAV control (n = 12) mice. See Fig. S for experimental scheme and Fig. S for HR excursion curves. h , i Time-dependent changes in MEE, EF and LVESD after FGF21 restoration in Fgf21 -/- mice (n = 31). See Fig. S c– . , k Energy expenditure (RER, VCO 2 , HEAT) by indirect calorimetry under basal conditions, after 48 hours of starvation, and after 24 hours of refeeding in the mouse groups as in . See Fig. S for experimental design and Fig. S10b–i for other analyses. For changes in energy expenditure parameters between the Fgf21 -/- and WT mice under similar conditions, see Fig. S a– . CN, control vector. SV, starvation. Left, excursion curves. Right, AUC of the respective curve. The AUC plot without a title indicates total (light + dark) AUC. l n = 18 per group. m n = 6 per group. l – p Changes in food intake and water consumption in the same groups and conditions as in – k . Data are means ± s.e.m.s; ( – p ) two-tailed unpaired Student’s t test; ( a – ) ordinary one-way ANOVA followed by Tukey’s test

Article Snippet: To establish a mouse model with mTOR inhibition, 8-week-old Fgf21 -/- mice were injected with Torin-1 (0.05 mg/mouse, i.p .) (MedChemExpress, USA) followed by a 12-h fast.

Techniques: Over Expression, Control, Plasmid Preparation, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: A two-strata energy flux system driven by a stress hormone prioritizes cardiac energetics

doi: 10.1038/s41392-025-02402-9

Figure Lengend Snippet: FGF21 deficiency induces a hypometabolic and mitochondrial hypoenergy state leading to cardiac dysfunction during fasting. a , Changes in mitochondrial metabolite/energy flux and key metabolic pathway enzymes involved in the TCA cycle, ETC and OXPHOS in Fgf21 -/- (n = 5-15) vs WT (n = 6-14) mice after 12 hours of fasting (fs) or 2 hours of rhFGF21 treatment, as analyzed by targeted cardiac energy metabolomics and qRT-PCR. See Fig. S for a summary heatmap. Transcriptomic and pathway enrichment in mitochondrial energy metabolism and cardiac function changes in Fgf21 -/- (n = 3) vs WT (n = 3) mice, with Reactome terms. For the GO-term and KEGG-term results, see Fig. , . For pathway enrichments in the Reactome term, GO term and KEGG term in Fgf21 -/- mice before and after rhFGF21 treatment, see Fig. S . For mitochondrial biogenesis, TCA cycle, ETC complexes I-IV, and OXPHOS, see Figs. S , S d, and S . For 24-h fasting effects, see Fig. S a– . d , e Significant pathway defects associated with striated muscle contraction and heart rate regulation in the hearts of Fgf21 -/- (n = 3) vs WT (n = 3) mice and pathway normalization after 2 hours of FGF21 treatment (n = 3). For cardiac conduction, blood vessel diameter maintenance, and blood pressure regulation, see Fig. S c–e and S . f Inhibiting the TCA cycle with Cpi-613 (1 mg/mouse, i.p .) reduced rhFGF21-promoted heart rate (HR) improvements in fasted Fgf21 -/- mice (same n = 6-16 mice per group). See Echo parameters in Fig. S . Data are means ± s.e.m.s; two-tailed unpaired Student’s t-test; a , , d – f ordinary one-way ANOVA followed by Tukey’s test. a , f images are generated in PowerPoint

Article Snippet: To establish a mouse model with mTOR inhibition, 8-week-old Fgf21 -/- mice were injected with Torin-1 (0.05 mg/mouse, i.p .) (MedChemExpress, USA) followed by a 12-h fast.

Techniques: Quantitative RT-PCR, Two Tailed Test, Generated

Journal: Signal Transduction and Targeted Therapy

Article Title: A two-strata energy flux system driven by a stress hormone prioritizes cardiac energetics

doi: 10.1038/s41392-025-02402-9

Figure Lengend Snippet: FGF21 promotes cardiac catabolic pathways for diverse metabolites to ensure cardiac energetic efficiency under stress. a , b Analysis of the expression of representative mitochondrial FAO pathway genes by qRT-PCR in the hearts of fasted Fgf21 -/- (n = 7-14) vs WT (n = 7-15) mice and with rhFGF21 treatment (n = 8-15). For transcriptomic enrichment of the FAO pathway, see Fig. S . For FAO under a 24-h fast, see Fig. S b, . For the metabolism of TG, phospholipids, and lipoproteins, see Fig. S d, . Effects of palmitate supplementation (2 mmol per mouse, i.p .) on the heart rate (HR) of fasted Fgf21 -/- (n = 11) vs WT (n = 10) mice. See Fig. S for other related comparisons. d , Changes in mitochondrial beta-hydroxybutyrate (BHB) ketolysis pathway determined by qRT-PCR in the hearts of fasted Fgf21 -/- (n = 7-15) vs WT (n = 7-14) mice and those with rhFGF21 treatment (n = 8-15). See Fig. S for transcriptomic enrichment of ketolysis and Fig. S19c for ketolysis after a 24-h fast. For utilization of other ketones, see Fig. S d, . f Effects of 1,3-butanediol monoester (BHB precursor, 10 mmol/mouse via tail vein) on HR in fasted Fgf21 -/- (n = 11) vs WT (n = 12) mice. See Fig. S for other related comparisons. g Effects of 1,3-butanediol monoester on EF in fasted Fgf21 -/- (n = 29) vs WT (n = 30) mice. See Fig. S for effects on CO. h Effects of 1,3-butanediol monoester on serum BHB levels in fasted Fgf21 -/- (n = 6) vs WT (n = 6) mice. See Fig. S for effects on serum FFA, TG, glucose, and total cholesterol. i Effects of cardiac Oxct1 knockdown (KD) by AAV-mediated shRNA (2.5 ×10 11 GC/mouse via tail vein) on rhFGF21-promoted HR improvement in fasted Fgf21 -/- mice (n = 10) vs those with control AAV (n = 11-12). See Fig. S for the knockdown efficiency and Fig. S20e, f for more detailed comparisons and Echo parameters. j , k Transcriptomic changes in branched-chain amino acid (BCAA) oxidation pathway in the hearts of fasted Fgf21 -/- (n = 3) vs WT (n = 3) mice and those with rhFGF21 treatment (n = 3). See Fig. S b, for detailed pathway changes under both 12-h and 24-h fasts, and Figs. S a, S d, e and S for amino acid metabolism and protein turnover. See Fig. S – for leucine supplementation effects. IV-CoA, isovaleryl-CoA; MB-CoA, 2-methylbutyryl-CoA; IB-CoA, isobutyryl-CoA. Data are means ± s.e.m.s; ( , f , i ) two-tailed unpaired Student’s t-test; ( b , , g , h , k ) ordinary one-way ANOVA followed by Tukey’s test. ( a , d , g ) Images are generated in PowerPoint

Article Snippet: To establish a mouse model with mTOR inhibition, 8-week-old Fgf21 -/- mice were injected with Torin-1 (0.05 mg/mouse, i.p .) (MedChemExpress, USA) followed by a 12-h fast.

Techniques: Expressing, Quantitative RT-PCR, Knockdown, shRNA, Control, Two Tailed Test, Generated

Journal: Signal Transduction and Targeted Therapy

Article Title: A two-strata energy flux system driven by a stress hormone prioritizes cardiac energetics

doi: 10.1038/s41392-025-02402-9

Figure Lengend Snippet: FGF21 promotes peripheral and transcardiac metabolic flux to meet cardiac energy needs during prolonged fasting. a , b Expression analysis of representative genes in hepatic ketogenesis pathway by qRT-PCR in Fgf21 -/- (n = 5) vs WT mice (n = 5), under basal, 24-h fast, and acute rhFGF21 treatment (n = 5) conditions. For the response to rhFGF21, see Fig. S . For the 12-h fast effects, see Fig. S . The bar symbols follow those in Fig. . For pathway changes in FFAs sourced from WAT lipolysis, see Fig. S a-S . Changes in serum beta-hydroxybutyrate (BHB) and FFAs in Fgf21 -/- (n = 7-12) vs WT mice (n = 8-12) after fasting (both 12-h and 24-h) and rhFGF21 treatment (n = 7-12). For serum glucose and TG, see Fig. S . d Effects of hepatic Hmgcs2 knockdown (KD) on rhFGF21-promoted improvement of HR in Fgf21 -/- mice subjected to a 24-h fast compared with control AAV (n = 6-9 per group). Right: AUC of HR excursion curves; see Fig. S b, for more details. For the Echo parameters, see Fig. S . e Changes of serum BHB levels upon hepatic Hmgcs2 KD and rhFGF21 treatment in Fgf21 -/- mice after a 24-h fast. f Effects of adipocyte-specific ablation of Fgfr1 ( Fgfr1 f/f ;Adipoq CreERT ) on the improvement of HR promoted by acute rhFGF21 treatment or AAV9-TBG -hFGF21 mediated overexpression ( hFGF21 OE ) in mice fasted for 24 hours (n = 11-12 per group). OE, overexpression. hFGF21 , human FGF21 coding sequence. Right: AUC analysis, in which star symbols for p -values without underlines indicate a comparison to Fgfr1 -ablated mice under normal conditions (baseline). See Fig. S for experimental scheme and S for the Echo parameters. g Changes of serum FFAs in Fgfr1 f/f ;Adipoq CreERT mice. h Effects of the ATGL inhibitor Atglistatin (1.42 mg/mouse, i.p .) on the rhFGF21-promoted improvement of HR in Fgf21 -/- mice fasted for 12 hours (n = 11-12 per group). See Fig. S for experimental scheme and notes, Fig. S28d for more comparisons, and Fig. S28e for the Echo parameters. i Effects of Atglistatin on the rhFGF21-promoted increases in serum FFAs in Fgf21 -/- mice fasted for 12 hours (n = 9-10 per group), from which the heart draws FFA fuel. Data are means ± s.e.m.s; ( d , g , h ) Two-tailed unpaired Student’s t-test; ( b , , e , f , i ) ordinary one-way ANOVA followed by Tukey’s test. (a) Image is generated in PowerPoint

Article Snippet: To establish a mouse model with mTOR inhibition, 8-week-old Fgf21 -/- mice were injected with Torin-1 (0.05 mg/mouse, i.p .) (MedChemExpress, USA) followed by a 12-h fast.

Techniques: Expressing, Quantitative RT-PCR, Knockdown, Control, Over Expression, Sequencing, Comparison, Two Tailed Test, Generated

Journal: Signal Transduction and Targeted Therapy

Article Title: A two-strata energy flux system driven by a stress hormone prioritizes cardiac energetics

doi: 10.1038/s41392-025-02402-9

Figure Lengend Snippet: A mechanistic model for the regulation of heart energy allocation by energy stress hormone FGF21. The heart is an unrelenting bioengine that relies on a robust and uninterrupted influx of energy substates. Under physiologically relevant stressors, cardiomyocyte-derived or endocrine FGF21 acts to maintain heart rate, contractility, and hemodynamic stability by activating a dual energy flux system. Systemically, FGF21 directly promotes lipolysis in white adipose depots, releasing free fatty acids (FFAs) for liver uptake or direct transcardiac uptake via intracardiac microvascular circulation. It also promotes hepatic fatty acid oxidation and subsequent ketogenesis, supplying ketones for intracardiac ketolysis during prolonged fasting or starvation. These interorgan substrate mobilization effects ensure intracardiac energy substrate sufficiency. Locally, FGF21 signaling promotes transcardiac and intracardiac flux of various substrates for oxidative utilization, as well as cardiac mitochondrial biogenesis and respiration (the TCA cycle, ETC and OXPHOS), ensuring intracardiac ATP sufficiency. These processes/effects are mediated by the LKB1–AMPK and mTOR pathways and occur secondary to FGF21’s systemic actions. By coordinating these dual fuel systems, FGF21 signaling prioritizes incessant, robust intracardiac ATP flux, and thereby, cardiac energetic efficiency, particularly under stress. Thus, FGF21 is the first-known signaling factor for prioritizing cardiac energy needs and functional efficiency via a novel two-strata flux system. The image is generated in PowerPoint

Article Snippet: To establish a mouse model with mTOR inhibition, 8-week-old Fgf21 -/- mice were injected with Torin-1 (0.05 mg/mouse, i.p .) (MedChemExpress, USA) followed by a 12-h fast.

Techniques: Derivative Assay, Functional Assay, Generated