Journal: bioRxiv

Article Title: Plasma membrane accessible cholesterol is regulated by ACC1 and lipid droplets

doi: 10.1101/2025.08.21.671640

Figure Lengend Snippet: Random insertion mutagenesis screens identify new cholesterol regulatory genes a-d , Plasma membrane (PM) cholesterol was measured by mNeonGreen-ALOD4 (mNG-ALOD4) staining followed by flow cytometry. Wild type (WT) HAP1 cells are compared to GRAMD1A, GRAMD1B, and GRAMD1C triple knockout HAP1 cells ( GRAMD1 TKO) either left untreated ( a ) or treated with 4 µM 25-hydroxycholesterol (25HC) for the times indicated ( b ). WT cells were treated with or without LXR inhibitor (LXRi; 1 µM GSK2033) for 16 hrs in 5% FBS and either left untreated ( c ) or treated with 25HC for the times indicated ( d ). e , Schematic showing cholesterol trafficking from the PM to the ER membrane where it binds to proteins including SCAP and ACAT1 or ACAT2 (ACAT). ACAT esterifies cholesterol with a fatty acid (FA), enabling it to be stored in lipid droplets (LD). Cholesterol is transported to the PM from the Golgi . f , Schematic of genetic screening pipelines. The mutant cell library was treated with either of NPC1i (1 μM U18666A) for 20 hrs or 4 μM 25HC for 6 hrs and then cells were stained with mNG-ALOD4. The top 10% of mNG-ALOD4 fluorescent cells were isolated for sequencing. g,h, Screen results showing enriched genes with an FDR-corrected p -value of less than 2E-4 for cells treated with NPC1i ( g ) or 25HC ( h ). IGTIOB is a measure of the inactivating potential of the mapped insertions, and circle size shows the number of insertions for each gene. i,j , mNG- ALOD4 flow cytometry analysis of PM cholesterol in WT or ACC1 KO HAP1 cells treated with or without 1 μM of NPC1i for 20 hrs ( i ) or WT and NPC1 KO HAP1 cells treated with or without ACC1i (30 μM Firsocostat) for 16 hrs ( j ). k-m , Cells were left untreated ( k ) or treated for 16 hrs with 30 µM ACC1i and then treated with 4 µM 25HC for the times indicated ( l and m ). The amount of mNG-ALOD4 bound to the PM was measured by flow cytometry. Data is normalized to cells not treated with 25HC for each condition, and each circle is the mean of three biological replicates; error bars are the standard deviation. Statistical tests compare two conditions at the same time point of 25HC treatment ( d and k-m ). Statistical significance was determined by Student’s t-test with a Welch’s correction ( a-d and j- m ) or an Ordinary one-way ANOVA ( i ). Exact p -values are reported in the Methods.

Article Snippet: Small molecules used in this study include: 25-hydroxycholesterol (25HC) (11097, Cayman Chemical), the LXR inhibitor GSK2033 (1 μM) (25443, Cayman Chemical), the NPC1 inhibitor U18666A (1 μM) (10009085, Cayman Chemical) the ACC1 inhibitor Firsocostat (also named GS-0976 and ND-630) (30 μM) (HY-16901, MedChemExpress), the ACC1 inhibitor CP-640186 (20 μM) (HY15259, MedChemExpress), the HMGCR inhibitor Lovastatin (10 μM) (10010338, Cayman Chemical), the ACAT1/2 inhibitor SZ58-035 (50 μM) (S9318, Millipore Sigma), the FASN inhibitor C75 (10 μM) (10005270, Cayman Chemical), the AMPK inhibitor Dorsomorphin dihydrochloride (2.5 μM) (HY-13418, MedChemExpress), the long-chain fatty acyl CoA synthetase (ACSL) inhibitor Triacsin-C (5 μM) (BMLEI218, Enzo), human adipose triglyceride lipase (ATGL) inhibitor NG-497 (50 μM) (HY-148756, MedChemExpress), Oleic acid (200 μM) (O1383,Millipore Sigma), the DGAT1 inhibitor T863 (20 μM) (SML0539, Sigma-Aldrich) and the DGAT2 inhibitor PF-06424439 (10 μM) (PZ0233, Sigma-Aldrich).

Techniques: Mutagenesis, Clinical Proteomics, Membrane, Staining, Flow Cytometry, Triple Knockout, Isolation, Sequencing, Standard Deviation

Journal: bioRxiv

Article Title: Plasma membrane accessible cholesterol is regulated by ACC1 and lipid droplets

doi: 10.1101/2025.08.21.671640

Figure Lengend Snippet: ACC1 functions independently of ACAT1/2 to maintain plasma membrane cholesterol levels. a , Fluorescence microscopy image showing wild-type (WT) 3T3 cells left untreated or treated with 0.3 mM MβCD:cholesterol or ACC1i (30 μM Firsocostat) and stained with mNG-ALOD4 and DAPI. Scale bar is 10 microns. b-d , Cell lines were left untreated ( b ) or treated with 30 µM Firsocostat ( c ) or 20 μM CP-640186 for 16 hrs ( d ) and then stained with mNG-ALOD4 and analyzed by flow cytometry. e , Cells were treated with 4 µM 25HC for 4 hrs and then media was replaced with PFO* for 30 minutes at 37°C before cell viability was measured with an XTT assay. f , Schematic of cholesterol ester synthesis. ACAT1 and ACAT2 (ACAT) enzymes transfer a fatty acid from acyl-CoA to cholesterol, forming cholesterol esters. g , Western blot (ACAT1) and PCR ( ACAT2 ) analysis of ACAT1/2 DKO clones. h , WT and ACAT1/2 DKO cell lines were treated with 4 μM 25HC for 6 hrs and the esterified cholesterol was quantified using the Amplex Red assay. i , j , Cells were left untreated ( i ) or treated for 16 hrs with ACC1i (30 μM Firsocostat) ( j ); cells were then treated with 4 µM 25HC for the times indicated and then the amount of mNG-ALOD4 bound to the PM was measured by flow cytometry. k , Western blot (ACAT1 and ACC1) or PCR ( ACAT2 ) analysis of ACC1/ACAT1/2 TKO clones . l , Cells were treated with 4 µM 25HC for the indicated times and then mNG-ALOD4 staining was measured by flow cytometry. m , mNG-ALOD4 flow cytometry analysis of PM cholesterol in WT and ACAT1/2 DKO HAP1 cells left untreated or treated with ACC1i (30 μM Firsocostat) for 16 hrs. n , Quantification of total cellular free (unesterified) cholesterol in WT HAP1 cells or ACC1 KO HAP1 cells treated with or without 4 μM 25HC for 8 hrs. Cells ( b , d , and m ) were grown in lipoprotein depleted serum media for 16 hours prior to treatments. For each condition ( i , j and l ), data is normalized to cells not treated with 25HC, and each data point represents the average of three biological replicates, with error bars denoting the standard deviation. Statistical significance was determined by a Student’s t-test with a Welch’s correction ( b , c , h - j , l and n ) or an Ordinary one-way ANOVA ( d , and m ). Exact p -values are found in the Methods.

Article Snippet: Small molecules used in this study include: 25-hydroxycholesterol (25HC) (11097, Cayman Chemical), the LXR inhibitor GSK2033 (1 μM) (25443, Cayman Chemical), the NPC1 inhibitor U18666A (1 μM) (10009085, Cayman Chemical) the ACC1 inhibitor Firsocostat (also named GS-0976 and ND-630) (30 μM) (HY-16901, MedChemExpress), the ACC1 inhibitor CP-640186 (20 μM) (HY15259, MedChemExpress), the HMGCR inhibitor Lovastatin (10 μM) (10010338, Cayman Chemical), the ACAT1/2 inhibitor SZ58-035 (50 μM) (S9318, Millipore Sigma), the FASN inhibitor C75 (10 μM) (10005270, Cayman Chemical), the AMPK inhibitor Dorsomorphin dihydrochloride (2.5 μM) (HY-13418, MedChemExpress), the long-chain fatty acyl CoA synthetase (ACSL) inhibitor Triacsin-C (5 μM) (BMLEI218, Enzo), human adipose triglyceride lipase (ATGL) inhibitor NG-497 (50 μM) (HY-148756, MedChemExpress), Oleic acid (200 μM) (O1383,Millipore Sigma), the DGAT1 inhibitor T863 (20 μM) (SML0539, Sigma-Aldrich) and the DGAT2 inhibitor PF-06424439 (10 μM) (PZ0233, Sigma-Aldrich).

Techniques: Clinical Proteomics, Membrane, Fluorescence, Microscopy, Staining, Flow Cytometry, XTT Assay, Western Blot, Clone Assay, Amplex Red Assay, Standard Deviation

Journal: bioRxiv

Article Title: Plasma membrane accessible cholesterol is regulated by ACC1 and lipid droplets

doi: 10.1101/2025.08.21.671640

Figure Lengend Snippet: Lipid droplet catabolism increases plasma membrane cholesterol. a , Plasma membrane (PM) cholesterol was measured by mNG-ALOD4 staining followed by flow cytometry in WT HAP1 cells treated with ACC1i (30 µM Firsocostat) for the indicated times. b , c , Microscopy images of HAP1 cells left untreated or treated with ACC1i (30 μM Firsocostat), and then stained with ACC1 monoclonal antibody ( b ) or 647-conjugated ACC1 polyclonal antibody ( c ), Bodipy 493/503 to measure lipid droplets (LDs) and DAPI (blue). Scale bar is 5 microns. d , Schematic showing that enzymes involved in the synthesis of fatty acyl-CoAs, which are then converted into triacylglycerol (TAG), cholesterol esters (CE), sphingolipids (SL) and phospholipids (PL). TAG and CE are the main components of the LD core (green oval). Inhibitors targeting enzymes in this process are shown in gray. e - g mNG-ALOD4 flow cytometry analysis of PM cholesterol (blue curves) and the corresponding fluorescence microscopy quantification of the number of LDs per cell (purple curves) in HAP1 cells grown in 5% LDS ( e ) or 5% FBS ( f , g ) and then treated with ACC1i (30 μM Firsocostat) ( e , f ) or ACSLi (5 μM Triacsin-C) ( g ). LDs were quantified by taking the mean number of LDs per cell in four fields of view with ∼75 cells per field. h , i , mNG-ALOD4 flow cytometry analysis of PM cholesterol in WT HAP1 cells treated with or without ACSLi (5 μM Triacsin-C) for 16 hrs in 5% LDS, followed by treatment with 4 μM 25HC ( h ) or 30 μM ACC1i ( i ) for the indicated times. j , k , LD quantification ( j ) and mNG-ALOD4 flow cytometry analysis of PM cholesterol ( k ) of WT HAP1 cells treated with or without 30 μM ACC1i or 5 μM ACSLi for 16 hrs, followed by 200 μM oleic acid treatment for 6 hrs in 5% FBS. Each flow cytometry data point ( e - i ) represents the mean of three biological replicates, with error bars indicating the standard deviation. Values were normalized to the untreated control at the corresponding time point. Statistical significance was determined by Ordinary one-way ANOVA ( a ) or Student’s t-test with a Welch’s correction ( h - k ). Exact p -values are found in the Methods.

Article Snippet: Small molecules used in this study include: 25-hydroxycholesterol (25HC) (11097, Cayman Chemical), the LXR inhibitor GSK2033 (1 μM) (25443, Cayman Chemical), the NPC1 inhibitor U18666A (1 μM) (10009085, Cayman Chemical) the ACC1 inhibitor Firsocostat (also named GS-0976 and ND-630) (30 μM) (HY-16901, MedChemExpress), the ACC1 inhibitor CP-640186 (20 μM) (HY15259, MedChemExpress), the HMGCR inhibitor Lovastatin (10 μM) (10010338, Cayman Chemical), the ACAT1/2 inhibitor SZ58-035 (50 μM) (S9318, Millipore Sigma), the FASN inhibitor C75 (10 μM) (10005270, Cayman Chemical), the AMPK inhibitor Dorsomorphin dihydrochloride (2.5 μM) (HY-13418, MedChemExpress), the long-chain fatty acyl CoA synthetase (ACSL) inhibitor Triacsin-C (5 μM) (BMLEI218, Enzo), human adipose triglyceride lipase (ATGL) inhibitor NG-497 (50 μM) (HY-148756, MedChemExpress), Oleic acid (200 μM) (O1383,Millipore Sigma), the DGAT1 inhibitor T863 (20 μM) (SML0539, Sigma-Aldrich) and the DGAT2 inhibitor PF-06424439 (10 μM) (PZ0233, Sigma-Aldrich).

Techniques: Clinical Proteomics, Membrane, Staining, Flow Cytometry, Microscopy, Fluorescence, Standard Deviation, Control

Journal: bioRxiv

Article Title: Plasma membrane accessible cholesterol is regulated by ACC1 and lipid droplets

doi: 10.1101/2025.08.21.671640

Figure Lengend Snippet: ACC1 inhibition triggers lipid droplet catabolism through ATGL. a , Schematic illustrating the formation of fatty acyl-CoAs from de novo synthesis of fatty acids (top) or from the breakdown of triacylglycerol (TAG) into diacylglycerol and a fatty acid by ATGL (bottom). ATGL localizes at the lipid droplet (LD) surface. b , Western blot analysis of ATGL KO HAP1 cells compared to a wild-type (WT) control. c , d , Quantification of LD numbers per cell ( c ) or LD area ( d ) in WT and ATGL KO cells. e - g Microscopy images ( e , f ) and quantification ( g ) of LDs in WT ( e ) and ATGL KO ( f ) HAP1 cells treated with or without ACC1i (30 μM Firsocostat) for 6 hrs. Cells were stained with Bodipy 493/503 and DAPI (blue). Scale bar is 5 microns. Each data point is the total number of LDs per cell in a single field of view, with ∼75 cells per field ( c and g ). h , i , mNG-ALOD4 flow cytometry analysis of PM cholesterol in WT and ATGL KO cells ( h ) or WT cells treated with or without ATGLi (50 μM NG-497) ( i ), followed by treatment with ACC1i (30 μM Firsocostat) for the indicated times. All experiments were carried out in cells pre-cultured for 16 hours in lipoprotein depleted serum. Data points represent the average of three biological replicates, with error bars indicating the standard deviation. All values were normalized to the untreated control ( h , i ). Statistical significance was determined by a Student’s t-test with a Welch’s correction ( c , d and g - i ). Exact p -values are found in the Methods.

Article Snippet: Small molecules used in this study include: 25-hydroxycholesterol (25HC) (11097, Cayman Chemical), the LXR inhibitor GSK2033 (1 μM) (25443, Cayman Chemical), the NPC1 inhibitor U18666A (1 μM) (10009085, Cayman Chemical) the ACC1 inhibitor Firsocostat (also named GS-0976 and ND-630) (30 μM) (HY-16901, MedChemExpress), the ACC1 inhibitor CP-640186 (20 μM) (HY15259, MedChemExpress), the HMGCR inhibitor Lovastatin (10 μM) (10010338, Cayman Chemical), the ACAT1/2 inhibitor SZ58-035 (50 μM) (S9318, Millipore Sigma), the FASN inhibitor C75 (10 μM) (10005270, Cayman Chemical), the AMPK inhibitor Dorsomorphin dihydrochloride (2.5 μM) (HY-13418, MedChemExpress), the long-chain fatty acyl CoA synthetase (ACSL) inhibitor Triacsin-C (5 μM) (BMLEI218, Enzo), human adipose triglyceride lipase (ATGL) inhibitor NG-497 (50 μM) (HY-148756, MedChemExpress), Oleic acid (200 μM) (O1383,Millipore Sigma), the DGAT1 inhibitor T863 (20 μM) (SML0539, Sigma-Aldrich) and the DGAT2 inhibitor PF-06424439 (10 μM) (PZ0233, Sigma-Aldrich).

Techniques: Inhibition, Western Blot, Control, Microscopy, Staining, Flow Cytometry, Cell Culture, Standard Deviation

Journal: bioRxiv

Article Title: Plasma membrane accessible cholesterol is regulated by ACC1 and lipid droplets

doi: 10.1101/2025.08.21.671640

Figure Lengend Snippet: Increasing lipid droplet abundance reduces plasma membrane cholesterol. a - c , Wild-type (WT) HAP1 cells were left untreated or treated with ACSLi (5 μM Triacsin-C), ATGLi (50 μM NG-497) or both for 16 hrs and then analyzed by microscopy to quantify lipid droplet (LD) numbers per cell ( a , b ) or by mNG-ALOD4 flow cytometry analysis to determine plasma membrane (PM) cholesterol levels ( c ). d , Schematic demonstrating that AMPK inactivates ACC1 and activates ATGL. Inhibitors for each enzyme are shown in gray. e , Western blot analysis of ACC1 phosphorylation in WT HAP1 cells left untreated or treated with AMPKi (2.5 μM Dorsomorphin), ACC1i (30 μM Firsocostat) or both. f - h WT HAP1 cells were left untreated or treated with 30 μM ACC1i, 2.5 μM AMPKi or both and then analyzed by microscopy to quantify LD numbers per cell ( f , g ) or by mNG-ALOD4 flow cytometry analysis to determine PM cholesterol levels ( h ). i , j , LD quantification ( i ) and flow cytometry analysis of PM cholesterol using mNG-ALOD4 staining ( j ) in ACAT1/2 DKO cells left untreated or treated with 2.5 μM AMPKi, 30 μM ACC1i or both. All cells were grown in lipoprotein depleted serum media for 16 hours prior to drug treatments. Lipid droplets are visualized with Bodipy 493:503 and nuclei are visualized with DAPI. Scale bars are 5 microns. Statistical significance was determined by an Ordinary one-way ANOVA ( b - c and g - j ). Each data point is the total number of LD per cell in a single field of view ( b , g and i ). Exact p -values are found in the Methods.

Article Snippet: Small molecules used in this study include: 25-hydroxycholesterol (25HC) (11097, Cayman Chemical), the LXR inhibitor GSK2033 (1 μM) (25443, Cayman Chemical), the NPC1 inhibitor U18666A (1 μM) (10009085, Cayman Chemical) the ACC1 inhibitor Firsocostat (also named GS-0976 and ND-630) (30 μM) (HY-16901, MedChemExpress), the ACC1 inhibitor CP-640186 (20 μM) (HY15259, MedChemExpress), the HMGCR inhibitor Lovastatin (10 μM) (10010338, Cayman Chemical), the ACAT1/2 inhibitor SZ58-035 (50 μM) (S9318, Millipore Sigma), the FASN inhibitor C75 (10 μM) (10005270, Cayman Chemical), the AMPK inhibitor Dorsomorphin dihydrochloride (2.5 μM) (HY-13418, MedChemExpress), the long-chain fatty acyl CoA synthetase (ACSL) inhibitor Triacsin-C (5 μM) (BMLEI218, Enzo), human adipose triglyceride lipase (ATGL) inhibitor NG-497 (50 μM) (HY-148756, MedChemExpress), Oleic acid (200 μM) (O1383,Millipore Sigma), the DGAT1 inhibitor T863 (20 μM) (SML0539, Sigma-Aldrich) and the DGAT2 inhibitor PF-06424439 (10 μM) (PZ0233, Sigma-Aldrich).

Techniques: Clinical Proteomics, Membrane, Microscopy, Flow Cytometry, Western Blot, Phospho-proteomics, Staining

Journal: bioRxiv

Article Title: Plasma membrane accessible cholesterol is regulated by ACC1 and lipid droplets

doi: 10.1101/2025.08.21.671640

Figure Lengend Snippet: ACC1 loss traps cholesterol at the plasma membrane and trigger de nova cholesterol synthesis in a mouse model. a , Western blot analysis of ACC1 and ACC2 proteins in primary hepatocytes isolated from Acc1/Acc2 flox/flox and Albumin-Cre- Acc1/Acc2 dLKO mice. b - d , Hepatic fatty acid synthesis rates ( b ), hepatic triacylglycerol (TAG) levels ( c ), and hepatic cholesterol ester (CE) levels ( d ) in Acc1/Acc2 flox/flox and Albumin-Cre- Acc1/Acc2 dLKO mice. e , Fluorescence microscopy images of primary hepatocytes stained with mNG-ALOD4 and DAPI. Scale bar is 10 microns. Quantification is shown in ( f ). g , h , Bubble plot showing differential gene expression from RNA sequencing in WT HAP1 cells. Cells were pre-cultured in lipoprotein depleted serum (LDS) media for 16 hours and then left untreated or treated for 6 hrs with HMGCRi (10 µM Lovastatin), ACC1i (30 μM Firsocostat), ACSLi (5 μM Triacsin-C) or FASNi (10 μM C75). Differential gene expression was computed by comparing each treatment to the untreated (LDS only) control. Bubble size shows the FDR-corrected p -value and bubble color shows the fold change (see legends) for each SREBF2 ( g ) and SREBP1 ( h ) target gene. SREBP1 target genes are ranked based on their enrichment in a ChIP-Seq dataset. i , Gene Set Enrichment Analysis from RNA sequencing data using the top 200 genes sorted by the FDR-corrected p -value. “nf” refers to not found. j - l , Hepatic sterol synthesis rate ( j ), hepatic cholesterol content ( k ), or fecal cholesterol measurement ( l ) in chow-fed Acc1/Acc2 flox/flox and Albumin-Cre- Acc1/Acc2 dLKO mice. m , Schematic showing a proposed model: ACC1 inhibits ATGL, blocking the breakdown of TAG to diacylglycerol (DAG) and fatty acids (FA). When ACC1 is inhibited (right), ATGL is activated, leading to TAG hydrolysis. Cell diagrams show that lipid droplets (LD) control cholesterol trafficking from the PM to the ER. When LDs are depleted (right), cholesterol becomes trapped at the PM, and SREBP2 drives the expression of cholesterol biosynthesis genes. Statistical significance was determined by a Student’s t-test with a Welch’s correction ( b - d, f and j - l ). Exact p -values are found in the Methods.

Article Snippet: Small molecules used in this study include: 25-hydroxycholesterol (25HC) (11097, Cayman Chemical), the LXR inhibitor GSK2033 (1 μM) (25443, Cayman Chemical), the NPC1 inhibitor U18666A (1 μM) (10009085, Cayman Chemical) the ACC1 inhibitor Firsocostat (also named GS-0976 and ND-630) (30 μM) (HY-16901, MedChemExpress), the ACC1 inhibitor CP-640186 (20 μM) (HY15259, MedChemExpress), the HMGCR inhibitor Lovastatin (10 μM) (10010338, Cayman Chemical), the ACAT1/2 inhibitor SZ58-035 (50 μM) (S9318, Millipore Sigma), the FASN inhibitor C75 (10 μM) (10005270, Cayman Chemical), the AMPK inhibitor Dorsomorphin dihydrochloride (2.5 μM) (HY-13418, MedChemExpress), the long-chain fatty acyl CoA synthetase (ACSL) inhibitor Triacsin-C (5 μM) (BMLEI218, Enzo), human adipose triglyceride lipase (ATGL) inhibitor NG-497 (50 μM) (HY-148756, MedChemExpress), Oleic acid (200 μM) (O1383,Millipore Sigma), the DGAT1 inhibitor T863 (20 μM) (SML0539, Sigma-Aldrich) and the DGAT2 inhibitor PF-06424439 (10 μM) (PZ0233, Sigma-Aldrich).

Techniques: Clinical Proteomics, Membrane, Western Blot, Isolation, Fluorescence, Microscopy, Staining, Gene Expression, RNA Sequencing, Cell Culture, Control, ChIP-sequencing, Blocking Assay, Expressing

Journal: Advanced Science

Article Title: Glycerol Kinase Drives Hepatic de novo Lipogenesis and Triglyceride Synthesis in Nonalcoholic Fatty Liver by Activating SREBP‐1c Transcription, Upregulating DGAT1/2 Expression, and Promoting Glycerol Metabolism

doi: 10.1002/advs.202401311

Figure Lengend Snippet: Knockdown of Gk in the liver attenuates HFD‐induced hepatic Srebp‐1c and lipogenic gene expression, hepatic steatosis, and hyperlipidemia. A–C) Hepatic Gk mRNA (A) and TG (B) levels in mice fed a high‐fat diet (HFD) for different periods of time, and Spearman correlation analysis between hepatic Gk mRNA and TG levels (C). D–P) Body weight (D), serum levels of glycerol (E), TG (F), NFFA (G), AST (H) and ALT (I), hepatic TG content (J), images of the liver tissue stained with H&E and Oil‐red O (K), as well as hepatic expression of Gk , lipogenic genes and Srebp‐1c at mRNA and protein levels (L‐P) in mice fed with HFD for 2 months followed by infection with adenoviruses expressing either Gk shRNA (shGk) or control sequence (shCon) for 2 weeks. n = 6‐8/group. Data are expressed as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 (unpaired two‐tailed Student's t test). Scale bar: 100 µm. Acc, acetyl‐CoA carboxylase; Acly, ATP citrate lyase; ALT, alanine transaminase; AST, aspartate transaminase; Dgat, diacylglycerol acyltransferase; Elovl, elongase of very long chain fatty acids; Fasn, fatty acid synthase; Gk, glycerol kinase; Gpat, glycerol‐3‐phosphate acyltransferase; H&E, hematoxylin and eosin; NEFA, nonesterified fatty acids; Scd, stearoyl‐coenzyme A desaturase; Srebp‐1c, sterol regulatory element binding protein‐1c; nSREBP‐1c, nuclear form of SREBP‐1c; pSREBP‐1c, precursor of SREBP‐1c; TG, triglyceride.

Article Snippet: To investigate the effect of GK on TG synthesis and the involvement of SREBP‐1c in hepatocytes, HepG2 cells were transfected with SREBP‐1c siRNA (siSREBP‐1c) or control siRNA (siCon), or treated with 10 μM NDI‐091143 (S8878, Selleck, USA), 10 μM Firsocostat (S8893, Selleck), 10 μM C75 (S9819, Selleck) for 12 h. The cells then were transfected with GK expression plasmid or control vector for 36 to determine intracellular TG levels.

Techniques: Knockdown, Gene Expression, Staining, Expressing, Infection, shRNA, Control, Sequencing, Two Tailed Test, Binding Assay

Journal: Advanced Science

Article Title: Glycerol Kinase Drives Hepatic de novo Lipogenesis and Triglyceride Synthesis in Nonalcoholic Fatty Liver by Activating SREBP‐1c Transcription, Upregulating DGAT1/2 Expression, and Promoting Glycerol Metabolism

doi: 10.1002/advs.202401311

Figure Lengend Snippet: Cholesterol and fatty acids stimulate GK expression in hepatocytes. A,D,G,H) The GK mRNA levels in Huh7 cells stimulated with/without 50 µM cholesterol (A), 400 µM palmitate (B), 100 nM insulin (G), or different concentrations of glucose (H) for the indicated times. B,C,E,F) The mRNA (B,E) and protein (C, F) levels of GK in Huh7 cells treated with different concentrations of cholesterol or palmitate for 20 and 48 h, respectively. I‐K) The EGR1 , SP1 , and GK mRNA levels in Huh7 cells transfected with EGR1 siRNA (siEGR1), SP1 siRNA (siSP1) and stimulated with 400 µM palmitate (PA). L‐N) The KLF5 , SP1 , and GK mRNA levels in Huh7 cells transfected with KLF5 siRNA (siEGR1), SP1 siRNA (siSP1) and stimulated with 50 µM cholesterol. n = 3. Data are expressed as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, compared with cells treated with control medium at the corresponding time points (unpaired two‐tailed Student's t test). GK, glycerol kinase; EGR1, early growth response 1; SP1, Sp1 transcription factor; KLF5, Krüppel‐like factor 5 .

Article Snippet: To investigate the effect of GK on TG synthesis and the involvement of SREBP‐1c in hepatocytes, HepG2 cells were transfected with SREBP‐1c siRNA (siSREBP‐1c) or control siRNA (siCon), or treated with 10 μM NDI‐091143 (S8878, Selleck, USA), 10 μM Firsocostat (S8893, Selleck), 10 μM C75 (S9819, Selleck) for 12 h. The cells then were transfected with GK expression plasmid or control vector for 36 to determine intracellular TG levels.

Techniques: Expressing, Transfection, Control, Two Tailed Test

Journal: Advanced Science

Article Title: Glycerol Kinase Drives Hepatic de novo Lipogenesis and Triglyceride Synthesis in Nonalcoholic Fatty Liver by Activating SREBP‐1c Transcription, Upregulating DGAT1/2 Expression, and Promoting Glycerol Metabolism

doi: 10.1002/advs.202401311

Figure Lengend Snippet: Overexpression of GK in hepatocytes promotes TG synthesis through upregulation of SREBP‐1c and its target lipogenic gene expression. A) The mRNA levels of GK and SREBP‐1c in Huh7 cells were transduced with adenoviruses expressing GK (Ad‐GK) for different time periods. B) Immunofluorescence staining of GK and SREBP‐1c in Huh7 cells transfected with GK expression plasmid (GK) or control vector. C) The mRNA levels of GK and SREBP‐1c in Huh7 cells infected with Ad‐GK or adenoviruses carrying control vector (Ad‐Con), or transfected with siRNA against SREBP‐1c (si SREBP‐1c ) followed by infection with Ad‐GK. D–F) The mRNA (D) and protein (E,F) levels of lipogenic genes in Huh7 cells transduced with Ad‐Con or Ad‐ GK . G) Metabolic flux analysis of de novo fatty acid synthesis by using [ 13 C]‐acetate in HepG2 cells transfected with control siRNA (siCon) or siSREBP‐1c followed by transfection with GK expression plasmid (GK) or control vector. H) Intracellular TG levels in HepG2 cells transfected with control siRNA (siCon) or siSREBP‐1c followed by transfection with GK expression plasmid (GK) or control vector. I) Intracellular TG levels in HepG2 cells treated with ACLY inhibitor NDI‐091143, ACC inhibitor firsocostat, or FASN inhibitor C75 followed by transfection with GK expression plasmid (GK) or control vector. n = 3. Data are expressed as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, compared to Huh7 cells without Ad‐GK transfection (unpaired two‐tailed Student's t ‐test). Abbreviations in this figure are the same as in Figure .

Article Snippet: To investigate the effect of GK on TG synthesis and the involvement of SREBP‐1c in hepatocytes, HepG2 cells were transfected with SREBP‐1c siRNA (siSREBP‐1c) or control siRNA (siCon), or treated with 10 μM NDI‐091143 (S8878, Selleck, USA), 10 μM Firsocostat (S8893, Selleck), 10 μM C75 (S9819, Selleck) for 12 h. The cells then were transfected with GK expression plasmid or control vector for 36 to determine intracellular TG levels.

Techniques: Over Expression, Gene Expression, Transduction, Expressing, Immunofluorescence, Staining, Transfection, Plasmid Preparation, Control, Infection, Two Tailed Test

Journal: Advanced Science

Article Title: Glycerol Kinase Drives Hepatic de novo Lipogenesis and Triglyceride Synthesis in Nonalcoholic Fatty Liver by Activating SREBP‐1c Transcription, Upregulating DGAT1/2 Expression, and Promoting Glycerol Metabolism

doi: 10.1002/advs.202401311

Figure Lengend Snippet: Knockdown of Gk in mouse liver or hepatocytes does not significantly affect the expression of Srebp‐1c and its target lipogenic genes. A–J) Hepatic Gk mRNA level (A), body weight (B), serum levels of glycerol (C), NEFA (D) and TG (E), liver TG content (F), as well as the expression of hepatic Srebp‐1c , lipogenic genes, and TG synthesis genes at mRNA and protein levels (G‐J) in mice infected with adenoviruses expressing Gk shRNA (shGK) or control sequence (shCon) for 2 weeks. n = 6/group. K–O) The mRNA levels of GK , SREBP‐1c , lipogenic genes, and TG synthesis genes in AML12 cells transfected with Gk siRNA (siGk) or control siRNA (siCon). n = 3. Data are expressed as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 (unpaired two‐tailed Student's t test). Abbreviations in this figure are the same as in Figure .

Article Snippet: To investigate the effect of GK on TG synthesis and the involvement of SREBP‐1c in hepatocytes, HepG2 cells were transfected with SREBP‐1c siRNA (siSREBP‐1c) or control siRNA (siCon), or treated with 10 μM NDI‐091143 (S8878, Selleck, USA), 10 μM Firsocostat (S8893, Selleck), 10 μM C75 (S9819, Selleck) for 12 h. The cells then were transfected with GK expression plasmid or control vector for 36 to determine intracellular TG levels.

Techniques: Knockdown, Expressing, Infection, shRNA, Control, Sequencing, Transfection, Two Tailed Test