Journal: bioRxiv

Article Title: Novel Role of AcylCoA:cholesterol acyltransferase 1 (ACAT1/SOAT1) in Diabetic Retinopathy

doi: 10.1101/2025.10.09.681484

Figure Lengend Snippet: A. Immunolabeling of retina sections from human donors with DR showed increased ACAT1/SOAT1 immunoreactivity in the photoreceptor outer segments (OS) and retinal pigment epithelial (RPE) cells, as compared with non-diabetic control donors. Donor sample information is provided in supplementary table 1. Scale bar = 50 µm. n=3. B. Vitrectomy tissues collected from patients with PDR showed an increased level of CE, as measured by ELISA. Donor sample information is provided in supplementary table 2. Data are presented as mean ± SEM; n=4-5; **p<0.01.

Article Snippet: Mice were treated with intraperitoneal injections of a specific ACAT1/SOAT1 inhibitor (K604, MedChem Express, 10 mg/Kg in PBS) or vehicle control (PBS) every 2 days.

Techniques: Immunolabeling, Control, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Novel Role of AcylCoA:cholesterol acyltransferase 1 (ACAT1/SOAT1) in Diabetic Retinopathy

doi: 10.1101/2025.10.09.681484

Figure Lengend Snippet: Ten-week old male diabetic Ins2 Akita mice were treated with PBS as vehicle or ACAT1/SOAT1 inhibitor, K604 (10mg/Kg, i.p.), every 2 days for 2 weeks. After 2 weeks of treatment, eyeballs and retinas were collected. A-B. Oil red O staining of frozen sections shows mild reactivity in the ganglion cell layer (GCL) and photoreceptor outer segments (OS) of the Ins2 Akita diabetic retina. Treatment with the ACAT1/SOAT1 inhibitor, K604, reduced this activity. Mean ± SEM; n=4-5; *p<0.05; ns: not significant. C-D. Detection of CE by filipin labeling of frozen retina sections shows prominent increases in CE formation in the ganglion cell (GCL), outer plexiform (OPL) and photoreceptor outer segment (OS) layers of the Ins2 Akita diabetic retina. Treatment with K604 prevented the CE increase. Scale bar = 40 µm; mean ± SEM; n=4; ****p<0.0001. E-F. Total cholesterol and CE levels were measured in retinas and plasma from non-diabetic control mice, Ins2 Akita diabetic mice, and K604-treated Ins2 Akita diabetic mice. This analysis showed upregulation of total cholesterol in plasma of the Ins2 Akita mice but not in retina. However, CE was increased in both retina and plasma of the Ins2 Akita mice. K604 treatment reduced retinal CE formation as well as preventing the increases in both cholesterol and CE in plasma. Data are presented as mean ± SEM; n=4-6; **p<0.01; ***p<0.001; ****p<0.0001; ns: not significant.

Article Snippet: Mice were treated with intraperitoneal injections of a specific ACAT1/SOAT1 inhibitor (K604, MedChem Express, 10 mg/Kg in PBS) or vehicle control (PBS) every 2 days.

Techniques: Staining, Activity Assay, Labeling, Clinical Proteomics, Control

Journal: bioRxiv

Article Title: Novel Role of AcylCoA:cholesterol acyltransferase 1 (ACAT1/SOAT1) in Diabetic Retinopathy

doi: 10.1101/2025.10.09.681484

Figure Lengend Snippet: A. Western blot analysis of retinas from 12-week diabetic Ins2 Akita mice showed marked increases in B. LDLR, C. ACAT1/SOAT1, D. TREM1, E. MCSF, F. VEGF, and G. TNF-α as compared with the WT non-diabetic controls. These increases were normalized by the treatment with K604 (10mg/Kg, ip) every two days from 10 to 12 weeks. Mean ± SEM, n=3-6; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. H-I . Increased immunoreactivity for ACAT1/SOAT1 was detected in retinal frozen sections from Ins2 Akita diabetic mice, especially in the RGC, OPL, and OS layers. The ACAT1 immunoreactivity was significantly reduced in retinas of Ins2 Akita diabetic mice treated with K604. Data are presented as mean ± SEM; n=3-6; Scale bar = 25 μm.

Article Snippet: Mice were treated with intraperitoneal injections of a specific ACAT1/SOAT1 inhibitor (K604, MedChem Express, 10 mg/Kg in PBS) or vehicle control (PBS) every 2 days.

Techniques: Western Blot

Journal: bioRxiv

Article Title: Novel Role of AcylCoA:cholesterol acyltransferase 1 (ACAT1/SOAT1) in Diabetic Retinopathy

doi: 10.1101/2025.10.09.681484

Figure Lengend Snippet: A-B. DHE imaging showed upregulation of superoxide formation in frozen sections from Ins2 Akita diabetic mice. This effect was blocked by the K604 treatment. n=4. C-D. Immunofluorescent labeling of frozen retina sections with 4-hydroxynonenal (4-HNE) and fluorescence intensity quantification showed increased 4-HNE levels in the ganglion cell layer (GCL), outer plexiform layer (OPL) and photoreceptor outer segments (OS) of the Ins2 Akita mice. K604 treatment blocked this effect. n=4. E-F. Immunolabeling of retina cross-sections showed increased GFAP immunoreactivity in Müller cells of the Ins2 Akita mice, which was blocked in the K604 treated mice. n=4. Scale bar=30 μm. G-K. Quantitative RT-PCR showed upregulation of Acat1/Soat1 , along with proinflammatory mediators – Mcsf1, Il1b, Tnfa , and Nos2 in Ins2 Akita diabetic mouse retinas, compared to non-diabetic controls. K604 treatment significantly inhibited these increases. n=4-6. All the data are presented as mean ± SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Article Snippet: Mice were treated with intraperitoneal injections of a specific ACAT1/SOAT1 inhibitor (K604, MedChem Express, 10 mg/Kg in PBS) or vehicle control (PBS) every 2 days.

Techniques: Imaging, Labeling, Fluorescence, Immunolabeling, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Novel Role of AcylCoA:cholesterol acyltransferase 1 (ACAT1/SOAT1) in Diabetic Retinopathy

doi: 10.1101/2025.10.09.681484

Figure Lengend Snippet: Ten-week-old male diabetic Ins2 Akita mice were treated with ACAT1/SOAT1 inhibitor, K604 (10 mg/Kg, ip), every 2 days for 2 weeks. After 2 weeks, retinas were collected for analysis A-B. Confocal imaging of retinal flat mounts labeled with the neuronal marker, RBPMS and quantification showed a significant decrease in RPBMS positive retinal ganglion cells in vehicle-treated Ins2 Akita diabetic retinas compared to the non-diabetic control retinas. This retinal ganglion cell loss was markedly reduced by the K604 treatment. n=5-6; scale bar=40 μm. Data are presented as mean ± SEM. C-D . Retinal function was measured by electroretinography (ERG). Both scotopic a-wave and scotopic b-wave were improved in K604-treated Ins2 Akita diabetic mice as compared with the vehicle treated diabetic mice. n=12–26. Data are presented as mean ± SEM. **p<0.01 non-diabetic versus vehicle-treated diabetic mice; p<0.01 vehicle-treated diabetic versus K604-treated diabetic mice. E . Measurement of visual acuity by optoMotry responses (OMR) showed a marked improvement in the K604-treated diabetic mice as compared with the vehicle-treated diabetic mice. n=10-13. Data are presented as mean ± SEM. *p<0.05; ***p<0.0001.

Article Snippet: Mice were treated with intraperitoneal injections of a specific ACAT1/SOAT1 inhibitor (K604, MedChem Express, 10 mg/Kg in PBS) or vehicle control (PBS) every 2 days.

Techniques: Imaging, Labeling, Marker, Control

Journal: bioRxiv

Article Title: Novel Role of AcylCoA:cholesterol acyltransferase 1 (ACAT1/SOAT1) in Diabetic Retinopathy

doi: 10.1101/2025.10.09.681484

Figure Lengend Snippet: Ten week old male diabetic Ins2 Akita mice were treated with ACAT1/SOAT1 inhibitor, K604 (10 mg/Kg, ip), every 2 days for 2 weeks. After the 2 weeks treatment, retinas were collected for analysis. A-B. Quantitative analysis shows diabetes-induced increases in leukocyte adhesion in Ins2 Akita retinal vessels, which were significantly prevented by K604 treatment as compared with vehicles. Data are presented as mean ± SEM. n=5-6; *p<0.05; **p<0.01; Scale bar=50 μm. C-D . Vascular leakage was visualized and quantified by measuring the fluorescence intensity of FITC-conjugated BSA. K604 treatment significantly prevented vascular leakage in Ins2 Akita mice as compared with vehicle controls. Data are presented as mean ± SEM. n=5-6; scale bar=300 μm; ****p<0.0001. E . K604 treatment significantly prevented diabetes-induced increases in acellular capillary formation as compared with the vehicle controls. Data are presented as mean ± SEM. n=6, scale bar=40 μm; ****p<0.001.

Article Snippet: Mice were treated with intraperitoneal injections of a specific ACAT1/SOAT1 inhibitor (K604, MedChem Express, 10 mg/Kg in PBS) or vehicle control (PBS) every 2 days.

Techniques: Fluorescence

Journal: bioRxiv

Article Title: Novel Role of AcylCoA:cholesterol acyltransferase 1 (ACAT1/SOAT1) in Diabetic Retinopathy

doi: 10.1101/2025.10.09.681484

Figure Lengend Snippet: Eight-months-old male Ins2 Akita diabetic mice were treated with ACAT1/SOAT1 inhibitor, K604 (10 mg/Kg, ip), every 2 days for 2 months. After the 2 months treatment, retinas were collected for analysis. Oil red O staining ( A, B ), filipin staining ( C, D ) and 4HNE staining ( E, F ) of frozen sections shows a strong reaction in areas of the ganglion cell layer (GCL) and photoreceptor outer segments (OS) of the DR retina, indicating lipid accumulation, CE formation, and oxidative stress, respectively. Treatment with the ACAT1/SOAT1 inhibitor, K604, significantly blocked these effects. Scale bar=25 μm. Data are presented as mean ± SEM; n=3-4; **p<0.01; ***p<0.001; ****p<0.0001. G-J. Western blot analysis of retinal samples from the Ins2 Akita diabetic mice showed marked increases in LDLR, ACAT1/SOAT1, and TREM1, as compared with the WT non-diabetic controls. These increases were normalized by treatment with K604. Mean ± SEM; n=3; *p<0.05; ***p<0.001; ****p<0.0001.

Article Snippet: Mice were treated with intraperitoneal injections of a specific ACAT1/SOAT1 inhibitor (K604, MedChem Express, 10 mg/Kg in PBS) or vehicle control (PBS) every 2 days.

Techniques: Staining, Western Blot

Journal: bioRxiv

Article Title: Novel Role of AcylCoA:cholesterol acyltransferase 1 (ACAT1/SOAT1) in Diabetic Retinopathy

doi: 10.1101/2025.10.09.681484

Figure Lengend Snippet: Eight-months old male Ins2 Akita diabetic mice were treated with ACAT1/SOAT1 inhibitor, K604 (10 mg/Kg, ip), every 2 days for 2 months. After 2 months, retinas were collected for analysis. A-B . Albumin staining of retina sections showed a strong reaction in areas of Isolectin B4-positive blood vessels in the diabetic retina, indicating vascular leakage. Treatment with ACAT1/SOAT1 inhibitor, K604, significantly blocked this effect. Scale bar=25 μm; data are presented as mean ± SEM; n=3-4; *p<0.05; ****p<0.0001. C . Measurement of visual acuity by optoMotry responses (OMR) showed a significant impairment in the vehicle-treated diabetic Ins2 Akita mice that was significantly blunted in the K604-treated diabetic mice. n=6-10. Data are presented as mean ± SEM; **p<0.01; ****p<0.0001. D-E . Electroretinography (ERG) showed improvement in both scotopic a-wave and scotopic b-wave in K604-treated diabetic mice as compared with the vehicle treated diabetic mice. n=6-10. Data are presented as mean ± SEM. **p<0.01 non-diabetic versus vehicle-treated diabetic mice; p<0.01 vehicle-treated diabetic versus K604-treated diabetic mice.

Article Snippet: Mice were treated with intraperitoneal injections of a specific ACAT1/SOAT1 inhibitor (K604, MedChem Express, 10 mg/Kg in PBS) or vehicle control (PBS) every 2 days.

Techniques: Staining