Fig 1.
(A) Total body weight, (B) lean mass, and (C) fat mass of ncls- or veh-treated mice fed on either an HFD or an NCD. (D) Representative MRI cross-sectional images showing the distribution of visceral and subcutaneous fat in the mice described in (A). (E) Quantitative analysis of the abdominal fat tissue volume (visceral and subcutaneous) by MRI. (F) Representative hematoxylin & eosin (H&E)-stained epididymal WAT sections from the mice described in (A). Scale bar, 100 μm. (G) Mean area of adipocytes from the H&E sections shown in (F). (H) Frequency distribution of adipocyte sizes from the H&E sections shown in (F). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Underlying data and method of statistical analysis are provided in S1 Data.
Fig 2.
Ncls promotes fat clearance and improves blood metabolic parameters in HFD mice.
(A) Representative H&E-stained sections of liver, BAT, and skeletal muscle (quadriceps) from mice treated for 7 wk with ncls or veh on either an HFD or an NCD. Scale bars, 100 μm. (B) TG contents in liver, BAT, and quadricep muscles of the mice described in (A). Blood metabolic parameters were determined after 6 wk of ncls or veh administration. (C) Cholesterol, (D) fasting plasma leptin, (E) fasting plasma insulin, and (F) fasting plasma glucose levels were shown as bar graphs. (G) Oral glucose tolerance test (GTT), (H) GSIS assay and (I) insulin tolerance test (ITT) were performed in mice with ncls or veh treatment on either an HFD or an NCD. Area under curve (AUC) was calculated and shown as bar graphs in the right panels. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Underlying data and method of statistical analysis are provided in S1 Data.
Fig 3.
Ncls restores energy expenditure and physical activity of HFD mice to the levels of NCD mice.
(A) Oxygen consumption (VO2) over a 48-h period of mice treated for 6 wk with ncls or veh on either an HFD or an NCD. (B) Averages of VO2 in light and dark cycles. (C) Energy expenditure (EE) of the mice described in (A) over a 48-h period. (D) Mean RER of the mice described in (A) during light and dark cycles. Whole body (E) glucose and (F) lipid oxidation rates of the mice described in (A). (G) Physical activity plots of the mice described in (A) over two light and dark cycles. (H) Mean ambulatory counts of the mice described in (A) during light and dark cycles. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Underlying data and method of statistical analysis are provided in S1 Data.
Fig 4.
Ncls targets skeletal muscle to up-regulate signature genes of slow-twitch fibers in HFD mice.
(A) Hierarchical clustering of the 1,532 DEGs (> 2-fold change) in liver, WAT, BAT, and quadricep muscles of NCD-veh, HFD-veh, and HFD-ncls mice. (B) Most of the ncls up-regulated genes in quadricep muscles of HFD mice were also highly expressed in NCD-veh mice. (C) Most of the ncls down-regulated genes in quadricep muscles of HFD mice were also expressed at a lower level in NCD-veh mice as compared with HFD-veh mice. The percentages of overlapping genes among the up- and down-regulated genes in HFD-ncls mice were indicated in (B) and (C). Gene ontology analyses of the overlapping (D) 150 up-regulated and (E) 86 down-regulated genes in quadricep muscles. (F) Expression patterns of the ncls up-regulated genes in the “muscle protein” category in NCD-veh, HFD-veh and HFD-ncls mice. Underlying data and method of statistical analysis are provided in S1 Data.
Fig 5.
Ncls promotes slow-twitch fiber formation and oxidative metabolism in the skeletal muscle of HFD mice.
(A) Relative mRNA expression of slow-twitch and fast-twitch fiber marker genes in quadricep muscles in ncls- or veh-treated mice on either an HFD or an NCD. The values of the NCD-veh mice were arbitrarily set as one. (B) Representative H&E staining (top panels) and Myh7 immunostaining (lower panels) of quadricep muscles from the mice described in (A). Oxidative slow-twitch fibers are indicated by arrows, and they are smaller in size and redder in color as compared to glycolytic fast-twitch fibers. Myh7-positive fibers are also indicated by arrows. Scale bar, 50 μm. (C) qRT-PCR analysis of the selected metabolic genes in quadricep muscles from all groups of mice. (D) Mitochondrial respiration rate of intact EDL muscle assayed on a Seahorse extracellular flux analyzer from all four groups of mice. Quantification of serum free fatty acid (FFA) (E) and TG (F) in ncls- or veh-treated mice. (G) Relative mRNA expression of Atgl gene in quadricep muscles, WAT, and BAT. (H) Core body temperatures of the mice described in (A). (I) Ucp2 mRNA and (J) UCP2 protein levels in quadriceps muscle of veh- and ncls-treated mice were evaluated by qRT-PCR and western blotting. The asterisk denotes an unspecific band in the western blot, and the UCP2 band is indicated by an arrow. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was included as a loading control. (K) Mitochondrial DNA copy numbers in muscle, BAT, liver, and WAT were determined by qPCR and shown as bar graphs. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Underlying data and method of statistical analysis are provided in S1 Data.
Fig 6.
Ncls enhances mitochondrial respiration and FAO in both murine and primary human myotubes.
Relative mRNA expression of selected metabolic genes in (A) murine myotubes (C2C12) and (B) primary human myotubes (36C15Q) after exposure to 0.1 mM PA ± 20 nM ncls for 48 h. The values of the PA-treated myotubes were arbitrarily set as one. (C) C2C12 and (D) 36C15Q myotubes were stained with BODIPY (green) for lipid droplets and DAPI (blue) for nuclei (40x magnification). Mitochondrial respiration was evaluated with a Seahorse extracellular flux analyzer. The OCR output for the basal rate (BR), proton leak (PL), ATP production (AP), MR, and spare respiratory capacity (SRC) of ncls-treated (E) C2C12 and (F) 36C15Q myotubes were shown as bar graphs. The OCR was normalized to the total protein per well. FAO profiles of (G) C2C12 and (H) 36C15Q myotubes were determined by a Seahorse extracellular flux analyzer. Myotubes were incubated in substrate-limited medium overnight to prime the myotubes for utilization of exogenous fatty acids and assayed on the following day with 0.125 mM PA. Etomoxir (Eto, 40 μM) was used to inhibit FAO and to confirm the assay specificity. Vertical dashed lines indicate the time points of oligomycin (Oligo, 1 μM), FCCP (1.6 μM), and Rotenone/Antimycin A (Ret/A, 1 μM) injection. The ECAR of (I) C2C12 and (J) 36C15Q myotubes were determined by a Seahorse analyzer after treatment with 0.1 mM PA ± 20 nM ncls for 48 h. Vertical dashed lines indicate the time points of oligomycin (1.5 μM), FCCP (1.5 μM), and 2-deoxyglucose (2-DG, 100 mM) injection. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Underlying data and method of statistical analysis are provided in S1 Data.
Fig 7.
Ncls activates the AMPK signaling pathway in vitro and in vivo.
Ncls treatment led to (A) increased phospho-AMPKα (pAMPKα), increased phospho-ACC2 (pACC2), and (B) enhanced FAO in PA-treated C2C12 myotubes. These effects were significantly reduced by treatment of Compound C (Comp. C), a specific AMPK inhibitor. Western blots of total AMPKα and ACC2 (A) were used as controls for equal loading. Ratios of phospho- to total- AMPKα and ACC2 were shown as bar graphs below the representative immunoblotting images of two independent experiments performed in duplicates. Vertical dashed lines indicate the time points of oligomycin (Oligo, 1 μM), FCCP (1.6 μM), and Rotenone/Antimycin A (Ret/A, 1 μM) injection (B). OCRs were determined by a Seahorse extracellular flux analyzer. **** p < 0.0001. (C) Protein levels of AMPKα and ACC2 were evaluated by western blotting in quadricep muscles, liver, WAT, and BAT from HFD-veh mice. GAPDH was included as a loading control. Ncls treatment led to increased pAMPKα and pACC2 specifically in (D) muscle, but not in (E) liver or (F) BAT from HFD mice. (G) Ratios of phospho- to total- AMPKα and ACC2 were shown as bar graphs. ** p < 0.01, *** p < 0.001. Underlying data and method of statistical analysis are provided in S1 Data.
Fig 8.
Biochemical basis for AMPK activation by ncls and the impact of ncls treatment on mitochondrial functions.
(A) Ncls treatment led to increased MMP in C2C12 myotubes with or without PA treatment. The membrane-permeant JC-1 dye was used to monitor MMP in myotubes subjected to indicated treatments. Both flow cytometry and fluorescence microscopy results were shown for comparison. (B) cAMP concentration and (C) ADP/ATP ratio were examined in both ncls- and veh-treated C2C12 myotubes with or without PA treatment. (D) Hydrogen peroxide (H2O2) concentration was determined by an Amplex Red based assay kit in ncls- and veh-treated C2C12 myotubes with or without PA treatment. Data are presented as mean ± standard error of the mean (SEM). * p < 0.05, ** p < 0.01, **** p < 0.0001. Underlying data and method of statistical analysis are provided in S1 Data.