Journal: bioRxiv

Article Title: Glucokinase activity suppresses hepatic cholesterol synthesis and triglyceride accumulation: A new model for the effects of the GKRP P466L common human variant

doi: 10.64898/2026.04.07.717049

Figure Lengend Snippet: A) Western blot analysis of GKRP and GCK in livers from L-GKRPKO mice expressing either human GKRP (hGKRP) or the P446L variant. Samples were collected from mice after an overnight fast followed by 4 hours of refeeding after 13 weeks of HFD feeding. HSP90 was used as a loading control. B-D) Body weight (B), percent fat mass (C), and liver mass (% body weight) of hGKRP- and P446L-expressing mice over 13 weeks of HFD feeding (n = 8 per group). E) Glucose tolerance test on hGKRP- and P446L-expressing mice after 9 weeks on HFD diet (n = 8 per group). F) Plasma insulin levels in mice from (E) at baseline (fasted) and 15 minutes after glucose injection. Samples that measured below the assay’s limit of detection (0.1 ng/mL) were set to 0.1 (marked with a dashed line on the graph). G-L) L-GKRPKO mice expressing hGKRP and hGKRP P446L were fed a HFD for 13 weeks (n = 8 for both groups) G) Hepatic glycogen content after an overnight fast and refeeding for 4 hours. H) Hepatic TAG after overnight fast and refeeding for 4 hours. I) Plasma cholesterol after an overnight fast. J) Plasma cholesterol after an overnight fast and refeeding for 4 hours. K) Plasma TAG from overnight fasted mice. L) Plasma TAG after overnight fast and refeeding for 4 hours. Data are represented as mean ± SEM. Statistical significance was determined using either 2-way ANOVA with either Šidák post hoc test (B, C, and E) or Fisher’s Least Significant Difference (F) or Student’s t test (D, G-L). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: For the triglyceride assay, 20 μL of 1% sodium deoxycholate was added to each well and the plate was incubated at 37 °C for 10 min. 200 μL of Infinity Triglyceride Reagent (Thermo Scientific TR22421) or Infinity Total Cholesterol Reagent (Thermo Fisher Scientific TR13421) was added and the plates were incubated at 37 °C for 10 min. Absorbance was measured at 500 nm and concentration was calculated via standard curve.

Techniques: Western Blot, Expressing, Variant Assay, Control, Clinical Proteomics, Injection

Journal: bioRxiv

Article Title: Glucokinase activity suppresses hepatic cholesterol synthesis and triglyceride accumulation: A new model for the effects of the GKRP P466L common human variant

doi: 10.64898/2026.04.07.717049

Figure Lengend Snippet: A) Volcano plot showing differentially expressed genes identified from RNA-sequencing analysis of livers from control and L-GCKKO mice fed a HCD for 1 week. Samples were obtained from mice that were fasted overnight followed by refeeding for 6 hours. (n = 3/4 control/KO). Red labels: genes upregulated in KO vs. control (FC ≥ 2, adjusted p value < 0.05, total = 44). Green labels: upregulated cholesterol synthesis genes (total = 16). Blue labels: genes downregulated in KO vs. control (FC ≤ −2, adjusted p value < 0.05, total = 15). B) Gene ontology analysis of differentially regulated genes. Labels in each bar represent the number of differentially regulated genes. C) mRNA levels of cholesterogenic genes in livers from control and L-GCKKO mice fed a HCD for 1 week (n = 9/8 control/KO). D-F) Control and L-GCKKO mice were fed a HCD for 1 week. Samples were obtained from mice that were fasted overnight followed by refeeding for 24 hours. (n=10/8 control/KO). D) Total hepatic cholesterol. E) D 2 O-labeled hepatic cholesterol. F) % of labeled cholesterol (of total cholesterol). Data are represented as mean ± SEM. Statistical significance was determined using Student’s t test ( C-F ). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: For the triglyceride assay, 20 μL of 1% sodium deoxycholate was added to each well and the plate was incubated at 37 °C for 10 min. 200 μL of Infinity Triglyceride Reagent (Thermo Scientific TR22421) or Infinity Total Cholesterol Reagent (Thermo Fisher Scientific TR13421) was added and the plates were incubated at 37 °C for 10 min. Absorbance was measured at 500 nm and concentration was calculated via standard curve.

Techniques: RNA Sequencing, Control, Labeling

Journal: bioRxiv

Article Title: Glucokinase activity suppresses hepatic cholesterol synthesis and triglyceride accumulation: A new model for the effects of the GKRP P466L common human variant

doi: 10.64898/2026.04.07.717049

Figure Lengend Snippet: A) Western blot analysis of GCK and HKII in livers from control, L-GCKKO, and HKII-overexpressing L-GCKKO mice after 1 week on HCD. Samples were obtained from mice that were fasted overnight followed by refeeding for 24 hours. HSP90 was used as a loading control. B) Glucose tolerance test on control, L-GCKKO, and HKII-overexpressing L-GCKKO mice after 3 days on HCD (n = 9 per group). C) Plasma insulin levels from mice in (B) at baseline (fasting) and 15 minutes after glucose infusion (n = 9 for all groups). Samples that measured below the assay’s limit of detection (0.1 ng/mL) were set to 0.1 (marked with a dashed line on the graph). D-I) Control, L-GCKKO, and HKII-overexpressing L-GCKKO mice fed a HCD for 1 week. Measurements were performed on mice that were fasted overnight, followed by refeeding for 24 hours. D) Hepatic glycogen content. E) Liver mRNA levels of Mlxipl and Pklr . F) Liver mRNA levels of cholesterogenic genes. (D-F control n = 10, L-GckKO n = 9, +HKII n = 10) G) Total hepatic cholesterol. H) D 2 O-labeled hepatic cholesterol. I) % of labeled cholesterol (of total cholesterol). (G-I control n = 7, L-GckKO n = 8, +HKII n = 8). Data are represented as mean ± SEM. Statistical significance was determined using either 2-way ANOVA with Tukey’s post hoc test (B-C) or 1-way ANOVA with Tukey’s post hoc test (D-I). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: For the triglyceride assay, 20 μL of 1% sodium deoxycholate was added to each well and the plate was incubated at 37 °C for 10 min. 200 μL of Infinity Triglyceride Reagent (Thermo Scientific TR22421) or Infinity Total Cholesterol Reagent (Thermo Fisher Scientific TR13421) was added and the plates were incubated at 37 °C for 10 min. Absorbance was measured at 500 nm and concentration was calculated via standard curve.

Techniques: Western Blot, Control, Clinical Proteomics, Labeling

Journal: Frontiers in Immunology

Article Title: NPC1 promotes HTNV replication by controlling innate immune response

doi: 10.3389/fimmu.2026.1811629

Figure Lengend Snippet: NPC1 disrupts cellular cholesterol distribution. (A) Wild-type and NPC1 knockout HeLa cells were treated with U18666A (10 µM) and HTNV (MOI = 10), cellular cholesterol levels were measured at 48 h post-infection. Non-treatment, U18666A treatment only or HTNV infection only were all parallel control. (B) Wild-type and NPC1 knockout HeLa cells were treated with cholesterol (10 µM) and HTNV (MOI = 1) or not, and viral genome copies within medium and cells of each group were quantified using qPCR. (C) The cellular cholesterol distribution of each group was visualized by Filipin staining following the same treatment as (A) . (D) The represented captures of Filipin staining for each group. WT: wild type HeLa cells, KO: NPC1 knockout HeLa cells. Mock+DMSO: cells were treated with the same amount of DMSO and without HTNV infection; Mock+U18666A: cells were treated with10 μM U18666A and without HTNV infection; HTNV+DMSO: cells treated with the same amount of DMSO and with HTNV infection for 48 hours (MOI=1); HTNV+U18666A: cells were treated with10 μM U18666A andwith HTNV infection for 48 hours (MOI=1).

Article Snippet: Wild-type and NPC1 knockout HeLa cells were grown on a six-well plate for 12 h, 10 μM U18666A and HTNV (MOI = 1) were added to the cells, and the cellular cholesterol level was measured using Tissue Total Cholesterol (TC) Content Assay Kit (APPLYGEN) at 48 h post-infection.

Techniques: Knock-Out, Infection, Control, Staining

Journal: Frontiers in Immunology

Article Title: NPC1 promotes HTNV replication by controlling innate immune response

doi: 10.3389/fimmu.2026.1811629

Figure Lengend Snippet: Diagram of NPC1 functioning in HTNV life cycle. HTNV infection triggers cellular cholesterol redistribution, which requires NPC1. Cholesterol extensively accumulated in late-endosome, thereby amplifying the innate immune response, when HTNV infected NPC1 defective cells. Consequently, NPC1 avoids abnormal endosomal cholesterol accumulation to mitigate innate immune response and promote HTNV replication.

Article Snippet: Wild-type and NPC1 knockout HeLa cells were grown on a six-well plate for 12 h, 10 μM U18666A and HTNV (MOI = 1) were added to the cells, and the cellular cholesterol level was measured using Tissue Total Cholesterol (TC) Content Assay Kit (APPLYGEN) at 48 h post-infection.

Techniques: Infection