Journal: JCI Insight

Article Title: microRNA-483 ameliorates hypercholesterolemia by inhibiting PCSK9 production

doi: 10.1172/jci.insight.143812

Figure Lengend Snippet: ( A – E ) Male and female C57BL/6 mice were administered a single dose of AAV8-PCSK9 (w/o 3′-UTR) or AAV8 PCSK9-3′-UTR (with 3′-UTR) via tail vein injection ( n = 8 in each group) and fed an HFD for 12 weeks before killing. The gross appearance of mouse serum ( A ); serum levels of total cholesterol and triglycerides ( B ) ; FPLC detection of VLDL, LDL, and HDL ( C ); and representative Oil-red O staining of en face aortae ( D ) and aortic roots ( E ) are shown (original magnification, ×6; scale bars: 0.5 mm). In B , D , and E , data are mean ± SEM from 6–8 mice per group. Normally distributed data were analyzed by 2-tailed Student’s t test with Welch correction between 2 groups. * P < 0.05 vs. AAV8-PCSK9. AAV8, adeno-associated virus 8; PCSK9, proprotein convertase subtilisin/kexin type 9; AAV8-PCSK9-3′-UTR, AAV8-based recombinant virus encoding mouse Pcsk9 mRNA encompassing its native 3′-UTR; HFD, high-fat diet; FPLC, fast protein liquid chromatography; VLDL, very LDL-cholesterol.

Article Snippet: PCSK9 levels in cell culture medium and serum were measured with a human or mouse PCSK9 ELISA Kit (R&D Systems) according to the manufacturer’s protocols.

Techniques: Injection, Staining, Virus, Recombinant, Fast Protein Liquid Chromatography

Journal: JCI Insight

Article Title: microRNA-483 ameliorates hypercholesterolemia by inhibiting PCSK9 production

doi: 10.1172/jci.insight.143812

Figure Lengend Snippet: ( A and D ) The animals were used as described in . The hepatic Pcsk9 mRNA levels ( A ), ELISA detection of serum levels of PCSK9 ( A ), and hepatic levels of miR-191, -222, -224, and -483 ( D ) are shown. ( B ) Previously reported (blue lines) or newly predicted (green lines) miRs that bind to the h PCSK9 -3′-UTR are shown. ( C and E ) HepG2 cells were transfected with pre–miR-222 mimic (222), pre–miR-224 mimic (224), pre–miR-483 mimic (483), pre–miR-191 mimic (191), pre–miR-1912 mimic (1912), pre–miR-1295b mimic (1295b), or scramble miR control (Ctrl). In C , cells were cotransfected with Luc-PCSK9-3′-UTR reporter. Luciferase activity was measured with pRL-TK activity as a transfection control. In E , protein levels of PCSK9 and LDLR were determined by Western blot analysis; α-tubulin was a loading control. In A and D , data are mean ± SEM from 6–8 mice per group. Normally distributed data were analyzed by 2-tailed Student’s t test with Welch correction between 2 groups. In C and E , data are mean ± SEM from 3–4 independent experiments. Non-normally distributed data were analyzed using Mann-Whitney U test between indicated group and control. * P < 0.05 vs. AAV8-PCSK9 or Ctrl. miR, microRNA; PCSK9, proprotein convertase subtilisin/kexin type 9; HepG2, human hepatocellular carcinoma; LDLR, LDL receptor.

Article Snippet: PCSK9 levels in cell culture medium and serum were measured with a human or mouse PCSK9 ELISA Kit (R&D Systems) according to the manufacturer’s protocols.

Techniques: Enzyme-linked Immunosorbent Assay, Transfection, Control, Luciferase, Activity Assay, Western Blot, MANN-WHITNEY

Journal: JCI Insight

Article Title: microRNA-483 ameliorates hypercholesterolemia by inhibiting PCSK9 production

doi: 10.1172/jci.insight.143812

Figure Lengend Snippet: ( A ) Bioinformatics prediction of miR-483-5p binding sites in the 3′-UTR of human and mouse PCSK9 mRNA. In B – G , HepG2 cells were transfected with pre–miR-483 mimic (pre-483) or anti–miR-483 (anti-483) for 24 hours. ( B and C ) mRNA and protein levels of PCSK9 and LDLR. ( D ) HepG2 cells transfected with pre-483 or anti-483 were cotransfected with Luc-PCSK9-3′-UTR (WT) or Luc-mutated PCSK9-3′-UTR (MT). Luciferase activity was measured with pRL-TK activity as a transfection control. ( E ) Ago-1 or Ago-2 immunoprecipitation was performed, and miRISCs-associated miR-483, PCSK9 , and CTGF mRNA levels were quantified by qPCR. ( F and G ) mRNA and protein levels of PCSK9 and LDLR in WT HepG2 and mPCSK9 HepG2 cells were transfected with pre-483 or anti-483. # LDLR in the same samples were detected in parallel in a separate gel ( G ). Data are mean ± SEM from 3–4 independent experiments. In B – G , non-normally distributed data were analyzed using Mann-Whitney U test between indicated group and control. * P < 0.05 vs. control. miR, microRNA; PCSK9, proprotein convertase subtilisin/kexin type 9; HepG2, human hepatocellular carcinoma; LDLR, LDL receptor; Luc-PCSK9-3′-UTR (WT), WT PCSK9 3′-UTR; Luc-mutated PCSK9-3′-UTR (MT), mutant PCSK9 3′-UTR; miRISCs, miRNA-induced silencing complexes; CTGF , connective tissue growth factor.

Article Snippet: PCSK9 levels in cell culture medium and serum were measured with a human or mouse PCSK9 ELISA Kit (R&D Systems) according to the manufacturer’s protocols.

Techniques: Binding Assay, Transfection, Luciferase, Activity Assay, Control, Immunoprecipitation, MANN-WHITNEY, Mutagenesis

Journal: JCI Insight

Article Title: microRNA-483 ameliorates hypercholesterolemia by inhibiting PCSK9 production

doi: 10.1172/jci.insight.143812

Figure Lengend Snippet: ( A – H ) HepG2 and mPCSK9 HepG2 cells were transfected with pre-483 or anti-483 as indicated. Fluorescent-labeled LDL was incubated with HepG2 and mPCSK9 HepG2 cells. LDL uptake was detected by flow cytometry ( A and B ) or confocal microscopy ( C and D ) (original magnification, ×20; scale bars: 10 μm). ( E and F ) Levels of PCSK9 in conditioned media were measured by ELISA and Western blot analysis. ( G and H ) HepG2 and mPCSK9 HepG2 cells were incubated with 1 μM atorvastatin for 24 hours. mRNA and protein levels of PCSK9 and LDLR were determined by qPCR and Western blot analysis. In MT HepG2 cells, # LDLR in the same samples were detected in parallel in a separate gel ( H ). Data are mean ± SEM from at least 4 independent experiments. In A , B , and F , non-normally distributed data were analyzed using Mann-Whitney U test between 2 groups. In C – E , normally distributed data were analyzed by 2-tailed Student’s t test with Welch correction between 2 groups. In G and H , non-normally distributed data were analyzed using Kruskal-Wallis test with Dunn’s multiple comparisons between indicated groups. * P < 0.05 vs. Ctrl or between 2 indicated groups. miR, microRNA; HepG2, human hepatocellular carcinoma; LDL-C, LDL-cholesterol; PCSK9, proprotein convertase subtilisin/kexin type 9.

Article Snippet: PCSK9 levels in cell culture medium and serum were measured with a human or mouse PCSK9 ELISA Kit (R&D Systems) according to the manufacturer’s protocols.

Techniques: Transfection, Labeling, Incubation, Flow Cytometry, Confocal Microscopy, Enzyme-linked Immunosorbent Assay, Western Blot, MANN-WHITNEY

Journal: JCI Insight

Article Title: microRNA-483 ameliorates hypercholesterolemia by inhibiting PCSK9 production

doi: 10.1172/jci.insight.143812

Figure Lengend Snippet: ( A ) Male and female C57BL/6 mice were fed an HFD or chow diet for 6 weeks. A single dose of AAV8-null (AAV-null) or AAV8-pri-miR-483 (AAV-483) was administered by tail vein injection at the end of week 2. ( B and C ) Hepatic miR-483-5p levels were determined by qPCR, protein levels of PCSK9, LDLR, and CTGF were detected by Western blot. # CTGF in the same samples were detected in parallel in a separate gel ( C ). ( D ) Ago-1 was immunoprecipitated from fixed liver tissue, and Ago1-associated miR-483-5p, Pcsk9 , and Ctgf mRNA levels were quantified by qPCR. ( E ) Total cholesterol levels measured by cholesterol assay. ( F ) Serum levels of VLDL, LDL, and HDL were determined by FPLC. ( G ) The correlations between hepatic miR-483-5p expression levels and serum levels of PCSK9 (left) or total cholesterol (right) are shown. ΔCT represents the difference between the cycle threshold of miR-483-5p and U6. The numbers of mice used are shown in . Data are mean ± SEM. In B , non-normally distributed data were analyzed using Kruskal-Wallis test with Dunn’s multiple comparisons between indicated groups. In C and D , non-normally distributed data were analyzed using Mann-Whitney U test. In E , normally distributed data were analyzed by 1-way ANOVA test with a Bonferroni’s post hoc test between 2 indicated groups. In G , the correlation analysis was assessed by the Pearson method. * P < 0.05. miR, microRNA; LDL-C, LDL-cholesterol; HFD, high-fat diet; AAV8, adeno-associated virus 8; PCSK9, proprotein convertase subtilisin/kexin type 9; LDLR, LDL receptor; CTGF, connective tissue growth factor; VLDL, very LDL; IDL, intermediate-density lipoprotein.

Article Snippet: PCSK9 levels in cell culture medium and serum were measured with a human or mouse PCSK9 ELISA Kit (R&D Systems) according to the manufacturer’s protocols.

Techniques: Injection, Western Blot, Immunoprecipitation, Cholesterol Assay, Expressing, MANN-WHITNEY, Virus

Journal: JCI Insight

Article Title: microRNA-483 ameliorates hypercholesterolemia by inhibiting PCSK9 production

doi: 10.1172/jci.insight.143812

Figure Lengend Snippet: ( A ) Male and female C57BL/6 mice were administered AAV8-PCSK9-3′-UTR WT (WT) or AAV8-PCSK9-3′-UTR with a deleted miR-483 binding site (ΔBS) together with AAV-miR-483 or AAV-null by tail vein injection ( n = 6–13 in each group). All mice were fed an HFD for 8–10 weeks. ( B ) Levels of Pcsk9 and Ldlr mRNA in mouse liver were determined by qPCR. ( C ) Protein levels of hepatic LDLR were detected by Western blot analysis. ( D – F ) Serum levels of total cholesterol, VLDL, LDL, and HDL are shown. ( G ) Serum levels of PCSK9 assessed by ELISA are shown. The numbers of mice used are shown in . Data are mean ± SEM. In B , normally distributed data was analyzed by 1-way ANOVA test with a Bonferroni’s post hoc test between 2 indicated groups. In C , D , and G , non-normally distributed data were analyzed using Mann-Whitney U test between 2 indicated groups. * P < 0.05 vs. WT. miR, microRNA; LDL-C, LDL-cholesterol; AAV8, adeno-associated virus 8; PCSK9, proprotein convertase subtilisin/kexin type 9; AAV8-PCSK9-3′-UTR, AAV8-based recombinant virus encoding mouse Pcsk9 mRNA encompassing its native 3′-UTR; HFD, high-fat diet; LDLR, LDL receptor; VLDL, very LDL.

Article Snippet: PCSK9 levels in cell culture medium and serum were measured with a human or mouse PCSK9 ELISA Kit (R&D Systems) according to the manufacturer’s protocols.

Techniques: Binding Assay, Injection, Western Blot, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Virus, Recombinant

Journal: American journal of physiology. Gastrointestinal and liver physiology

Article Title: Retatrutide Improves Steatohepatitis in an Accelerated Mouse Model of Diet-Induced Steatohepatitis with a Fructose Binge

doi: 10.1152/ajpgi.00164.2025

Figure Lengend Snippet: For 31 days, female C57BL/6N mice were fed either chow diet and regular drinking water (Control), western diet and regular drinking water (Western Diet), western diet and drinking water with added fructose and sucrose (Western Diet + F/S), or western diet and drinking water with added fructose and sucrose plus a final fructose binge (10 mg/g body weight) 6 hours before sacrifice (Western Diet + F/S + Binge) (n=12 per group). (A) Absolute liver weight. (B) Liver weight-to-body weight ratio. (C) Plasma alanine aminotransferase content. (D) Hepatic triglyceride content. (E) Hepatic cholesterol content. (F-H) Hepatic gene expression of (F) Cxcl-1 , (G) Saa-1 , and (H) Saa-2 . (I) Hepatic collagen content. (J-K) Representative liver sections after (J) hematoxylin and eosin staining (bar size = 100 μ m) and (K) after Oil Red O staining (bar size = 100 μ m). Results are expressed as mean ± s.e.m. P values are determined by 1-way ANOVA, corrected for multiple comparisons using statistical hypothesis testing by Holm-Šídák (A-B), or Kruskal-Wallis with Dunn’s post hoc test (C-I). * P <0.05. ALT, alanine aminotransferase; Cxcl-1 , chemokine ligand-1; Saa-1 , serum amyloid A1; Saa- 2, serum amyloid A2.

Article Snippet: Hepatic cholesterol levels were measured using the Cholesterol Liquid Reagents Kit (Pointe Scientific, Canton, MI).

Techniques: Control, Western Blot, Clinical Proteomics, Gene Expression, Staining

Journal: American journal of physiology. Gastrointestinal and liver physiology

Article Title: Retatrutide Improves Steatohepatitis in an Accelerated Mouse Model of Diet-Induced Steatohepatitis with a Fructose Binge

doi: 10.1152/ajpgi.00164.2025

Figure Lengend Snippet: Female C57BL/6N mice were fed over 31 days either with chow diet (n=5 per group) and regular drinking water or western diet with added fructose and sucrose in the drinking water and a final fructose binge 6 hours prior to sacrifice (n=15 per group) with or without retatrutide treatment (30 nmol/kg body weight) on days 15, 18, 21, 24, 26, 28, and 30. (A) Absolute liver weight. (B) Liver weight-to-body weight ratio. (C) Plasma alanine aminotransferase content. (D) Hepatic triglyceride content. (E) Hepatic cholesterol content. (F-I) Hepatic gene expression of (F) Cxcl-1 , (G) Saa-1 , and (H) Saa-2 . (I-J) Representative liver sections after (I) hematoxylin and eosin staining (bar size = 100 μ m) and (J) after Oil Red O staining (bar size = 100 μ m). Results are expressed as mean ± s.e.m. P values are determined by 1-way ANOVA, corrected for multiple comparisons using statistical hypothesis testing by Holm-Šídák (A-E), or Kruskal-Wallis with Dunn’s post hoc test (F-H). * P <0.05. ALT, alanine aminotransferase; Cxcl-1 , chemokine ligand-1; Saa-1 , serum amyloid A1; Saa- 2, serum amyloid A2.

Article Snippet: Hepatic cholesterol levels were measured using the Cholesterol Liquid Reagents Kit (Pointe Scientific, Canton, MI).

Techniques: Western Blot, Clinical Proteomics, Gene Expression, Staining