acid oxidation detection  (Elabscience Biotechnology)

Journal: Advanced Science

Article Title: Asprosin‐FABP5 Interaction Modulates Mitochondrial Fatty Acid Oxidation through PPARα Contributing to MASLD Development

doi: 10.1002/advs.202415846

Figure Lengend Snippet: Asprosin induces lipid accumulation in hepatocytes. a) Primary hepatocytes were treated with different concentrations of asposin recombinant protein (r‐Asprosin, 20, 50, 100 ng mL −1 ) for 24 h. n = 6 in each group. b,d) Representative images of Oil Red O and BODIPY staining in primary hepatocytes and HepG2 cells. Cells were transfected with an asprosin overexpression plasmid for 24 h, followed by stimulation with 250 µM free fatty acids (FFA) for another 24 h. n = 5, 6 in each group. c) Detection of TC, TG content in primary hepatocytes. n = 6 in each group. e) Quantitative PCR was performed to determine the hepatic mRNA levels of genes related to fatty acid metabolism. n = 5, 6 in each group. f,g) Effects of asprosin knockdown on lipid accumulation in primary hepatocytes induced by 250 µM FFA, evidenced by reduced Oil Red O and BODIPY staining. n = 6 in each group. h) Detection of TC, TG content in primary hepatocytes. n = 6 in each group. i) BODIPY staining in HepG2 cells. j) Detection of TC, TG content in HepG2 cells. n = 6 in each group. Scale bar for Oil red O staining: 20 µm or 50 µm, for BODIPY staining: 10 µm or 20 µm. Statistical analysis was performed with one‐way ANOVA. * p < 0.05 versus NC, ** p < 0.01 versus NC, # p < 0.05 versus FFA (250 µmol/L), ## p < 0.01 versus FFA (250 µmol/L).

Article Snippet: FAO enzyme activities were quantitatively assessed using fatty acid oxidation detection reagent (#FDV‐0033, Funakoshi, Japan).

Techniques: Recombinant, Staining, Transfection, Over Expression, Plasmid Preparation, Real-time Polymerase Chain Reaction, Knockdown

Journal: Advanced Science

Article Title: Asprosin‐FABP5 Interaction Modulates Mitochondrial Fatty Acid Oxidation through PPARα Contributing to MASLD Development

doi: 10.1002/advs.202415846

Figure Lengend Snippet: Transcriptome analysis reveals asprosin enrichment pathway. a–c) RNA sequencing conducted on liver tissues from mice fed an HFHFHC diet and treated with either AAV‐NC (control) or AAV‐Asprosin (asprosin overexpression). Top 15 pathways identified through KEGG enrichment analysis showing significant alterations. Heatmap depicting the expression levels of genes involved in fatty acid metabolism, demonstrating differential expression between groups. d,e) Gene Set Enrichment Analysis (GSEA) analysis of PPAR signaling pathway, fatty acid degradation pathway, indicating shifts in gene expression profiles related to lipid metabolism in response to asprosin modulation. f) Western blot detection of PPARα and CPT1A protein levels in liver. n = 6 in each group. g) Hepatic mRNA levels of genes related to lipid metabolism. n = 5–8 in each group. h) HepG2 cells underwent FAOBlue staining to assess β‐oxidation capacity. n = 5 in each group. i) Triple immunofluorescence (IF) staining for Asprosin (red), PPARα (green), and nuclei (DAPI, blue) in HepG2 cells. n = 6 in each group. j) Co‐localization analysis of PPARα with asprosin. Scale bar: 5 µm. Statistical analysis was performed with one‐way ANOVA. ** p < 0.01 versus HFHFHC+AAV‐NC. HFHFHC, high fat, high cholesterol, high fructose.

Article Snippet: FAO enzyme activities were quantitatively assessed using fatty acid oxidation detection reagent (#FDV‐0033, Funakoshi, Japan).

Techniques: RNA Sequencing, Control, Over Expression, Expressing, Quantitative Proteomics, Gene Expression, Western Blot, Staining, Immunofluorescence

Journal: Advanced Science

Article Title: Asprosin‐FABP5 Interaction Modulates Mitochondrial Fatty Acid Oxidation through PPARα Contributing to MASLD Development

doi: 10.1002/advs.202415846

Figure Lengend Snippet: AAV‐shAsprosin enhances the effects of fenofibrate in HFCDAA‐fed mice. a) Western blot detection of PPARα and CPT1A protein levels in liver. n = 6 in each group. b–o) Statistical images of the oblique diameter of the right lobe of the liver detected by Doppler ultrasound; The body weight, liver weight, and LW/BW of C57BL/6J mice; The TC and TG content of livers; Plasma biochemical tests. Statistical analysis was performed with one‐way ANOVA. n = 6‐8 in each group. ALT, aspartate aminotransferase; AST, alanine aminotransferase; AKP, alkaline phosphatase; TC, total cholesterol; TG, triglyceride; HDL‐C, high‐density lipoprotein cholesterol; LDL‐C, low‐density lipoprotein cholesterol; LW/BW, liver weight/body weight; ND, normal diet; HFCDAA, high fat, methionine choline deficiency diet. Statistics of Oil Red O positive areas, NAFLD activity score (NAS), quantitative of F4/80 and CD68 positive area in the liver are shown. n = 6, 7 in each group. g) C57BL/6 J mice were fed on HFCDAA and treated with the indicated AAVs. After C57BL/6 J mice were fed on HFCDAA for 4 weeks, mice were injected with vehicle or fenofibrate (100 mg kg −1 ) for 4 weeks. Representative images, the oblique diameter of the right lobe of the liver detected by Doppler ultrasound, Oil Red O, H&E, and Masson's trichrome. n = 6–8 in each group. h) Representative images of liver biopsies stained for F4/80 immunofluorescence and CD68 immunohistochemistry. p,q) Schematic diagram of primary hepatocytes extracted for subsequent experiments. Primary hepatocytes, isolated from the livers of treated mice, underwent FAOBlue staining to assess β‐oxidation capacity, and BODIPY staining to evaluate lipid deposition. The statistics of quantification were evaluated by FAOBlue staining and BODIPY staining. n = 4 in each group. HFCDAA, high fat, methionine choline deficiency diet. r) q‐PCR was performed to use the hepatic mRNA levels of genes related to fatty acid metabolism. n = 5,6 in each group. Scale bar for Oil Red O, H&E, Masson's trichrome staining, and CD68 immunohistochemistry staining: 50 µm, for FAOBlue and BODIPY staining: 10 µm, Scale bar for F4/80 immunofluorescence staining: 100 µm. Statistical analysis was performed with one‐way ANOVA. ** p < 0.01,*** p < 0.001 versus AAV‐NC+ND; # p < 0.05; ## p < 0.01 versus AAV‐NC+HFCDAA; && p < 0.01 versus AAV‐NC+Fenofibrate+HFCDAA; ns, not significant. ND, normal diet; HFCDAA, high fat, methionine choline deficiency diet.

Article Snippet: FAO enzyme activities were quantitatively assessed using fatty acid oxidation detection reagent (#FDV‐0033, Funakoshi, Japan).

Techniques: Western Blot, Clinical Proteomics, Activity Assay, Injection, Staining, Immunofluorescence, Immunohistochemistry, Isolation

Journal: Aging (Albany NY)

Article Title: Systemic lipolysis promotes physiological fitness in Drosophila melanogaster

doi: 10.18632/aging.204251

Figure Lengend Snippet: bmm overexpression increases mitochondrial biogenesis and oxidative metabolism. ( A , B ) Level of 3-hydroxybutyrate (log 2 abundance) in da-GAL4/+ vs. da-GAL4>UAS-bmm flies. n=5 replicates, and each replicate contained 20 flies. FDR=0.0008 for bmm overexpression vs. control females, and FDR=0.13 for males. ( C , D ) β-oxidation rate measurement using fatty acid oxidation direct detection reagent, FAOblue (10 μM for 1 hour incubation). Each group consists of n=7-9 replicates, and each replicate sample was extracted from 20 flies. Data are shown as mean±SEM. Statistical analysis was carried out by two-tailed Student t -test in ( A – D ). ( E , F ) Oxygen consumption rate measurements of isolated mitochondria from da-GAL4/+ and da-GAL4>UAS-bmm flies, when stimulated by indicated reagents. Data are shown as mean±SEM and normalized by mitochondria protein. Statistical analysis was carried out by two-way ANOVA. n=5-6 replicates, and each replicate contained mitochondria extracted from 50 flies. Mal/Pyr: malate + Pyruvate; AA: antimycin A. ( G , H ) Quantification of mitochondrial DNA copy number in da-GAL4/+ vs. da-GAL4>UAS-bmm flies. Data are shown as fold change of mitochondrial Cytb DNA normalized to nuclear histone DNA. n=6-11 replicates, and each replicate was extracted from 10 flies. ( I , J ) Quantification of mitochondrial content in da-GAL4/+ vs. da-GAL4>UAS-bmm flies. n=4 replicates for females, and n=6 replicates for males. Mitochondria were isolated from 50 flies for each replicate, and protein content was quantified by BCA analysis. ( K , L ) MitoTracker intensity measurement of da-GAL4/+ vs. da-GAL4>UAS-bmm flies to assess mitochondrial mass. n=4 replicates for females, and n=6 replicates for males with 50 flies for each replicate. ( M , N ) Mitochondrial UPR genes mRNA expression level measurement in da-GAL4/+ vs. da-GAL4>UAS-bmm flies. n=7-11 replicates. ( O , P ) H 2 O 2 level measurement in da-GAL4/+ vs. da-GAL4>UAS-bmm flies. n=6 replicates. Data are shown as mean±SEM. Statistical analysis was carried out by two-tailed Student t -test. See also .

Article Snippet: β-oxidation rate measurement was carried out using fatty acid oxidation direct detection reagent, FAOblue (Funakoshi, Cat#: FDV-0033) at 10 μM for 1 hour [ ].

Techniques: Over Expression, Control, Incubation, Two Tailed Test, Isolation, Expressing