Journal: bioRxiv

Article Title: Tip60-mediated Rheb acetylation links palmitic acid with mTORC1 activation and insulin resistance

doi: 10.1101/2023.08.18.553816

Figure Lengend Snippet: (A) Heat map of the contents of acyl-CoAs and acyl-carnitines in fish myocytes treated with PA, OA or LA for 12h. The relative fold change for each factor in each sample is represented as the relative average increase (red) or decrease (blue) (n=3). (B) Oxygen consumption rate (OCR) traces as measured by seahorse XF 24 Flux Analyzer in fish myocytes treated with PA, OA or LA for 12h (n=3). (C) Relative mRNA levels of mitochondrial fatty acid β oxidation-related genes were analyzed by quantitative PCR in the muscle of fish fed CON or PO diet (n=4). (D and E) Relative mRNA levels of mitochondrial fatty acid β oxidation-related genes were measured by quantitative PCR in fish myocytes (B) and C2C12 myotubes (C) with control or PA treatment for 12 h (n=3). (F) Schematic representation of the main cellular pathways involved in mitochondrial fatty acid β oxidation. (G) The activities of mTORC1 and AKT were analyzed via immunoblotting in fish with intraperitoneal injection of control dsRNA or dsRNA targeting CPT1B for 36 h (n=6). (H) The activities of mTORC1 and AKT were assayed by immunoblotting in fish myocytes and C2C12 myotubes treated with control or etomoxir treatment in the presence or absence of PA for 12 h (n=3). (I) The activities of mTORC1 and AKT were assayed by immunoblotting in fish myocytes and C2C12 myotubes treated with control or perhexiline maleate treatment in the presence or absence of PA for 12 h (n=3). (J) The activity of mTORC1 signaling was examined by immunoblotting in C2C12 myotubes transfected with control siRNA or siRNA against CPT1B in the presence or absence of PA (n=3). (K) The activity of mTORC1 signaling was measured by immunoblotting in C2C12 myotubes transfected with control siRNA or siRNA against CPT2 in the presence or absence of PA (n=3). (L) Insulin-stimulated glucose uptake was detected by 2-DG uptake assays in fish myocytes under control or perhexiline maleate in the presence or absence of PA for 12 h (n=3). (M) AKT phosphorylation levels in fish myocytes and C2C12 myotubes were tested by immunoblotting (n=3). Cells were pretreated with control or perhexiline maleate treatment in the presence or absence of PA for 12 h, and then stimulated with insulin for 5 min. (N) AKT phosphorylation levels in fish myocytes were tested by immunoblotting (n=3). Cells were pretreated with control or etomoxir treatment in the presence or absence of PA for 12 h, and then stimulated with insulin for 5 min. (O) AKT phosphorylation levels in C2C12 myotubes were measured by immunoblotting (n=3). Cells were transfected with control siRNA or siRNA against CPT1B and pretreated with control or PA treatments for 12 h, and then stimulated with insulin for 5 min. The results are presented as the mean ± SEM and were analyzed using independent t -tests (* p < 0.05, ** p < 0.01, *** p < 0.001). See also .

Article Snippet: To inhibit mitochondrial fatty acid β oxidation, 50 μM etomoxir or 25 μM perhexiline maleate was added to the culture medium of fish primary myocytes or C2C12 myotubes for 12 h in the presence or absence of PA. For BMS-303141 treatment, BMS-303141 (Med Chem Express, #HY-16107, USA) was dissolved in DMSO (Solarbio, China) to obtain 25 mM stock solutions.

Techniques: Real-time Polymerase Chain Reaction, Control, Western Blot, Injection, Activity Assay, Transfection

Journal: bioRxiv

Article Title: Tip60-mediated Rheb acetylation links palmitic acid with mTORC1 activation and insulin resistance

doi: 10.1101/2023.08.18.553816

Figure Lengend Snippet: (A) Acetyl-CoA levels were measured in the muscle of fish fed CON or PO diet (n = 6). (B) Acetyl-CoA levels in fish myocytes were measured in the presence of the indicated concentrations of PA for 12 h (n = 3). (C) Acetyl-CoA levels were measured in fish myocytes under control or PA treatment with or without perhexiline maleate for 12 h (n = 3). (D) Acetyl-CoAs labeling pattern from fish myocytes treated with [U- 13 C 16 ]-labeled palmitate (n = 5). (E) Schematic representation of the main cellular pathways involved in ACLY-governed citrate transport and ACSS2-mediated acetyl-CoA production. (F) The activities of mTORC1 and AKT were assayed via immunoblotting in fish with intraperitoneal injection of control dsRNA or dsRNA targeting ACLY for 36 h (n=6). (G) The activity of mTORC1 was tested by immunoblotting in fish myocytes with control or BMS-303141 treatment in the presence or absence of PA for 12 h (n=3). (H) The activity of mTORC1 signaling was analyzed by immunoblotting in C2C12 myotubes transfected with control siRNA or siRNA against ACLY under control or PA treatment (n=3). (I) The activity of mTORC1 signaling was measured by immunoblotting in fish myocytes and C2C12 myotubes with the indicated concentrations of sodium acetate under control or PA treatment for 12 h (n=3). (J) The activity of mTORC1 signaling was measured by immunoblotting in fish myocytes and C2C12 myotubes under control or perhexiline maleate treatment with or without sodium acetate addition in the presence or absence of PA for 12 h (n=3). (K) Immunoblotting of Rheb acetylation and S6K phosphorylation in fish myocytes and C2C12 myotubes treated with the indicated concentrations of PA for 12 h (n=3). (L) Immunoblotting of Rheb acetylation in the muscle of fish fed CON or PO diet (n = 5). (M) Immunoblotting of Rheb acetylation and S6K phosphorylation in fish myocytes and C2C12 myotubes treated with control or perhexiline maleate treatment in the presence or absence of PA for 12 h (n=3). (N) Immunoblotting of Rheb acetylation and S6K phosphorylation in fish myocytes and C2C12 myotubes treated with the indicated concentrations of sodium acetate in the presence or absence of PA for 12 h (n=3). (O) Insulin-stimulated glucose uptake was detected by 2-DG uptake assays in fish myocytes under control or perhexiline maleate treatment with or without sodium acetate addition in the presence or absence of PA for 12 h (n=3). (P) AKT phosphorylation levels were tested by immunoblotting in fish myocytes (n=3). Cells were pretreated under control or perhexiline maleate treatment with or without sodium acetate addition in the presence or absence of PA for 12 h, and then stimulated with insulin for 5 min. (Q) AKT phosphorylation levels were detected by immunoblotting in fish myocytes (n=3). Cells were pretreated with control or BMS-303141 treatment in the presence or absence of PA for 12 h, and then stimulated with insulin for 5 min. The results are presented as the mean ± SEM and were analyzed using independent t -tests (* p < 0.05, ** p < 0.01, *** p < 0.001) and Tukey’s tests (bars bearing different letters are significantly different among treatments ( p < 0.05)). See also .

Article Snippet: To inhibit mitochondrial fatty acid β oxidation, 50 μM etomoxir or 25 μM perhexiline maleate was added to the culture medium of fish primary myocytes or C2C12 myotubes for 12 h in the presence or absence of PA. For BMS-303141 treatment, BMS-303141 (Med Chem Express, #HY-16107, USA) was dissolved in DMSO (Solarbio, China) to obtain 25 mM stock solutions.

Techniques: Control, Labeling, Western Blot, Injection, Activity Assay, Transfection

Journal: Advanced Healthcare Materials

Article Title: 3D Microtumors Representing Ovarian Cancer Minimal Residual Disease Respond to the Fatty Acid Oxidation Inhibitor Perhexiline

doi: 10.1002/adhm.202404072

Figure Lengend Snippet: MRD 3D microtumors reveal specific drug responses toward fatty acid oxidation inhibitors. (A,B) Cell viability changes of naïve and MRD 3D microtumors compared to a DMSO control after a 10‐day treatment with (A) 40 µ m etomoxir; (B) 4 µ m perhexiline ( n = 25–93 3D microtumors from 3 to 7 independent experiments). (C,D) Heatmaps of differentially expressed genes (DEGs) upregulated in etomoxir‐resistant MRD 3D microtumors composed of (C) OVCAR5 and (D) OVCAR‐5/RFP. (E) Confocal images of MRD 3D microtumors (3D MT) at D10 stained with the FAO marker CPT1A (yellow), phalloidin (pink), DAPI (blue). Scale bar = 20 µm. (F) Heatmap of DEGs upregulated in perhexiline‐resistant MRD 3D microtumors composed of OVCAR5. (G) Confocal images showing the DEGs upregulated in perhexiline‐resistant MRD 3D microtumors composed of OVCAR5 at day 10: AKR1B10 (green, top left panel), AKR1C2 (green, top right panel), FASN (green, bottom left panel), SCD (green, bottom right panel). The cells were also stained with phalloidin (pink), DAPI (blue). Scale bar = 20 µm.

Article Snippet: Etomoxir sodium salt (Stratech, #S8244‐SEL) was dissolved in DMSO to prepare a 40 m m stock solution, which was diluted in cell medium to a final concentration of 40 μ m . Perhexiline (Cambridge Bioscience, #CAY16982) was dissolved in DMSO to prepare a 4 m m perhexiline stock solution, which was diluted in cell medium to a final concentration of 4 μ m . DMSO was added to cell medium at a 1:1000 volume ratio for the control group.

Techniques: Control, Staining, Marker

Journal: Cell Death Discovery

Article Title: Potential role of lipophagy impairment for anticancer effects of glycolysis-suppressed pancreatic ductal adenocarcinoma cells

doi: 10.1038/s41420-024-01933-4

Figure Lengend Snippet: A PANC-1 cells were cultured in glucose or low-glucose medium with or without perhexiline (10 µM) for 120 h prior to measuring the total intracellular ATP content. Data are normalized against the levels in PANC-1 cells cultured in glucose medium without perhexiline. Data are presented as mean ± SD for experiments performed in triplicate. Statistical analysis is based on two-way ANOVA followed by Tukey’s test for multiple comparisons. Staining of LDs (green); nuclei are shown in blue. Scale bar, 20 µm. Higher-magnification images are shown on the right side ( B , C ) or the downside ( D ). PANC-1 cells were cultured in glucose or low-glucose medium with or without perhexiline (10 µM) for 48 h ( B ), and PANC-1, PK-1, and KLM-1 cells were cultured in glucose or low-glucose medium with or without SP-2509 (1 and 10 µM) for 48 h ( C ). PANC-1 cells were transfected with si-control or si-LSD1-1 for 24 h and then the medium was replaced. Transfected cells were cultured in low-glucose medium with SP-2509 (10 µM), OG-L002 (50 µM), iadademstat (50 µM), T-3775440 (50 µM), or control agent for 48 h. The cell lysates were also subjected to western blotting for LSD1 and β-actin antibodies ( D ). E PANC-1 cells were cultured in low-glucose medium with or without SP-2509 (10 µM) or OG-L002 (50 µM) for 24 h, 48 h, 72 h and 96 h. LDs are shown in green and nuclei are shown in blue. Scale bar, 20 µm. Higher-magnification images are shown on the right side. *** P < 0.001; ns not significant.

Article Snippet: SP-2509 (Merck, Darmstadt, Germany), OG-L002 (Selleckchem), T-3775440 HCl (Selleckchem), DCA (Tokyo Kasei Corp., Tokyo, Japan), perhexiline maleate salt (Cayman Chemical Company, Ann Arbor, MI, USA), and oligomycin (Sigma-Aldrich) were dissolved in DMSO (Nacalai Tesque Inc., Kyoto, Japan).

Techniques: Cell Culture, Staining, Transfection, Control, Western Blot

Journal: Cell Death Discovery

Article Title: Potential role of lipophagy impairment for anticancer effects of glycolysis-suppressed pancreatic ductal adenocarcinoma cells

doi: 10.1038/s41420-024-01933-4

Figure Lengend Snippet: A PANC-1, PK-1, and KLM-1 cells were cultured in glucose or low-glucose medium with or without chloroquine (CQ; 10 µM) for 48 h. Lipid droplets (LDs) are shown in green and nuclei are shown in blue. Scale bar, 20 µm. Higher-magnification images are shown on the right side. B , C PANC-1 cells were cultured in low-glucose medium with or without perhexiline (10 µM), SP-2509 (10 µM) or OG-L002 (50 µM) for 48 h. LDs are shown in green, lysosomes (Lyso) are shown in red and nuclei are shown in blue. Higher-magnification images are shown on the right side. Scale bar, 20 µm. B The cell lysates were subjected to western blotting for LC3, p62, and β-actin antibodies. For immunofluorescence, LC3 is shown in green and nuclei are shown in blue. Scale bar, 20 µm ( C ).

Article Snippet: SP-2509 (Merck, Darmstadt, Germany), OG-L002 (Selleckchem), T-3775440 HCl (Selleckchem), DCA (Tokyo Kasei Corp., Tokyo, Japan), perhexiline maleate salt (Cayman Chemical Company, Ann Arbor, MI, USA), and oligomycin (Sigma-Aldrich) were dissolved in DMSO (Nacalai Tesque Inc., Kyoto, Japan).

Techniques: Cell Culture, Western Blot, Immunofluorescence

Journal: bioRxiv

Article Title: Direct expression of CPT1a enables a high throughput platform for the discovery of CPT1a modulators

doi: 10.1101/2024.12.15.628575

Figure Lengend Snippet: IC 50 of a) etomoxir, b) perhexiline, c) chlorpromazine. A series of concentrations of each compound was used accordingly. We used the same amount of human CPT1a pellet solution with a protein concentration of 0.67 μg/μL, adding 2 μL of protein in each group.

Article Snippet: Two previously reported CPT1 inhibitors, etomoxir sodium salt (Cayman Chemical, Cat. #11969) and perhexiline (AK Scientific, Cat. #X4611), were used to validate the potential of this assay as a platform to screen for novel CPT1 inhibitors.

Techniques: Protein Concentration