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: bioRxiv

Article Title: Dietary depletion of glutamine is atheroprotective

doi: 10.64898/2026.03.06.710174

Figure Lengend Snippet: ( A ) Experimental design. Apoe -/- male mice were fed WD and received drinking water with no additives (Ctrl) or water supplemented with glutamine (Gln), or alpha-ketoglutarate (AKG) for 8 weeks. ( B ) Oil red O analysis of plaque burden in the aorta with ( C ) representative images of aortas from male mice. Scale bar: 2 mm. ( D ) Plaque area at 7 locations across the aortic root from the first appearance of the aortic valves in (top) male and (bottom) female mice. ( E ) Representative images of aortic root plaques from male and female mice in control (Ctrl) and glutamine (Gln) group. Scale bar: 200 μm. Black dashed line marks representative plaques boundaries. ( F ) Total plaque volume in the aortic root, calculated as the area under the curve from (D). ( G ) Sirius red analysis of plaque collagen content. ( H ) Necrotic core size analysis at the point of peak stenosis. ( I ) Plasma cholesterol. ( J ) Glutamine plasma concentration normalized to sex matched controls. (B, F, G, I, J) Data analyzed using one-way ANOVA with Dunnett’s correction or (H) Kruskal Wallis with Dunn’s correction for post-hoc analysis with N ≥ 13. Error bars represent mean ± SEM or (H) median ± IQR. *** P < 0.001; ** P = 0.001 to 0.01; * P = 0.01 to 0.05; ns, not significant.

Article Snippet: Plasma cholesterol was analyzed, using Infinity Cholesterol Liquid Stable Reagent kit (Thermo Scientific; cat#: TR13421) and Data-Trol A, Abnormal Control Serum (Thermo Scientific; cat#: TR41001), as an internal standard, according to the manufacturer’s instructions.

Techniques: Control, Clinical Proteomics, Concentration Assay

Journal: bioRxiv

Article Title: Gene dosage imbalance disrupts systemic metabolism in the Dp16 Down syndrome mouse model

doi: 10.64898/2026.01.13.699318

Figure Lengend Snippet: A-B) Overnight fasting insulin, blood glucose, serum triglyceride, cholesterol, non-esterified free fatty acids (NEFA), and β-hydroxybutyrate (ketone) in male (A) and female (B) Dp16 and WT mice. Sample size for male mice (WT = 15; Dp16 = 12) and female mice (WT = 14; Dp16 = 15). C-F) Impaired glucose tolerance as determined by the glucose tolerance test (GTT) in male (C) and female (E) Dp16 mice compared to WT controls. Impaired insulin sensitivity as determined by the insulin tolerance test (ITT) in male (D) and female (F) Dp16 compared to WT controls. Sample size for male mice (WT = 15; Dp16 = 12) and female mice (WT = 14; Dp16 = 15). G-H) Impaired triglyceride clearance in response to lipid gavage as determined by the lipid tolerance test (LTT) in male (G) and female (H) Dp16 relative to WT controls. Sample size for male mice (WT = 10; Dp16 = 14) and female mice (WT = 15; Dp16 = 15). I-J) Pooled mouse sera from male (I) and female (J) Dp16 and WT mice were fractionated by fast protein liquid chromatography (FPLC), and the triglyceride and cholesterol content of each fraction was quantified. Fractions corresponding to very-low density lipoprotein (VLDL), low-density lipoprotein (LDL), intermediate-density lipoprotein (IDL), and high-density lipoprotein (HDL) are indicated. All data are presented as mean ± SEM. * P <0.05; ** P <0.01; *** P <0.001; **** P <0.0001. For all tolerance tests, data were analyzed by 2-way ANOVA with Sidek post hoc tests.

Article Snippet: Following tissue extraction, total cholesterol content was quantified using the Infinity Cholesterol Reagent kit (Thermo Fisher Scientific, Middletown, VA) according to the manufacturer’s instructions.

Techniques: Fast Protein Liquid Chromatography

Journal: bioRxiv

Article Title: Gene dosage imbalance disrupts systemic metabolism in the Dp16 Down syndrome mouse model

doi: 10.64898/2026.01.13.699318

Figure Lengend Snippet: Quantification of hepatic TAG and DAG (by TLC method), and cholesterol (by infinity assay kit) levels in chow-fed Dp16 male (A-C) and female mice (D-F) and their corresponding WT controls. Sample size: male WT = 10 and Dp16 = 30; female WT = 15 and Dp16 = 10.

Article Snippet: Following tissue extraction, total cholesterol content was quantified using the Infinity Cholesterol Reagent kit (Thermo Fisher Scientific, Middletown, VA) according to the manufacturer’s instructions.

Techniques:

Journal: bioRxiv

Article Title: Gene dosage imbalance disrupts systemic metabolism in the Dp16 Down syndrome mouse model

doi: 10.64898/2026.01.13.699318

Figure Lengend Snippet: A-B) Overnight fasting insulin, blood glucose, serum triglyceride, cholesterol, non-esterified free fatty acids (NEFA), and β-hydroxybutyrate (ketone) in male (A) and female (B) Dp16 and WT mice on HFD. Sample size for male mice (WT = 15; Dp16 = 12) and female mice (WT = 14; Dp16 = 14). C-F) Exacerbated glucose intolerance as determined by the glucose tolerance test (GTT) in male (C) and female (E) Dp16 compared to WT controls on HFD. Exacerbated insulin resistance as determined by the insulin tolerance test (ITT) in male (D) and female (F) Dp16 compared to WT controls. Sample size for male mice (WT = 15; Dp16 = 12) and female mice (WT = 14; Dp16 = 14). G-H) The rate of triglyceride clearance in response to lipid gavage as determined by the lipid tolerance test (LTT) in male (G) and female (H) Dp16 and WT mice. Sample size for male mice (WT = 15; Dp16 = 12) and female mice (WT = 14; Dp16 = 14). I-J) Pooled mouse sera from male (I) and female (J) Dp16 and WT mice were fractionated by fast protein liquid chromatography (FPLC), and the triglyceride and cholesterol content of each fraction was quantified. Fractions corresponding to very-low density lipoprotein (VLDL), low-density lipoprotein (LDL), intermediate-density lipoprotein (IDL), and high-density lipoprotein (HDL) are indicated. All data are presented as mean ± SEM. * P <0.05; ** P <0.01; *** P <0.001; **** P <0.0001. For all tolerance tests, data were analyzed by 2-way ANOVA with Sidek post hoc tests.

Article Snippet: Following tissue extraction, total cholesterol content was quantified using the Infinity Cholesterol Reagent kit (Thermo Fisher Scientific, Middletown, VA) according to the manufacturer’s instructions.

Techniques: Fast Protein Liquid Chromatography