Journal: Molecular Metabolism

Article Title: Hmgcs2-mediated ketogenesis modulates high-fat diet-induced hepatosteatosis

doi: 10.1016/j.molmet.2022.101494

Figure Lengend Snippet: Ketogenesis activation through HMGCS2 overexpression improves hepatosteatosis in HFD-induced NAFLD mice. (A) Representative image of Ad- GFP and Ad- HMGCS2 mouse livers. (B) Liver weights at 3 weeks post-virus injection (Ad- GFP , n = 7; Ad- HMGCS2 , n = 9). (C) H&E and anti-Plin2 IHC staining of liver sections. Scale bar = 100 μm. Boxes indicate regions of higher magnification. (D) Histological liver fat quantification (n = 5/group). (E) NAFLD activity score (NAS) (n = 5/group). (F) Liver triglyceride concentrations of Ad- GFP and Ad- HMGCS2 mice (Ad- GFP , n = 5; Ad- HMGCS2 , n = 7). Gene expression analysis of (G) lipid accumulation ( Pparg , Fsp27 , Plin2 ), (H) lipid synthesis ( Srebp1c , Acc1 , Fasn ) and (I) lipid oxidation ( Ppara , Cpt1a , Scad , Mcad , Lcad ) (Ad- GFP , n = 7; Ad- HMGCS2 , n = 9) markers. Data are represented as mean ± SEM. Statistical analysis was performed by student's t -test. ∗ P ≤ 0.05; ∗∗ P ≤ 0.01; ∗∗∗ P ≤ 0.001.

Article Snippet: Lipid droplets were visualized by staining with hematoxylin and eosin (H&E) or immunohistochemistry with anti-Perilipin 2 (Plin2) antibody (1:150, NB110-40877; Novus Biologicals) and the VECTASTAIN Elite ABC reagent (PK-6100; Vector Laboratories).

Techniques: Activation Assay, Over Expression, Virus, Injection, Immunohistochemistry, Activity Assay, Gene Expression

Journal: Molecular Metabolism

Article Title: Hmgcs2-mediated ketogenesis modulates high-fat diet-induced hepatosteatosis

doi: 10.1016/j.molmet.2022.101494

Figure Lengend Snippet: NAFLD mice exhibit impaired fasting-induced ketogenesis due to abnormal Hmgcs2 expression in the liver. (A) Hematoxylin & Eosin (H&E) and anti-perilipin 2 (Plin2) immunohistochemical (IHC) staining of liver sections from healthy mice fed standard chow (SC) (22% kcal fat, 55% kcal carbohydrates, 23% kcal protein) and NAFLD mice fed high-fat diet (HFD) (45% kcal fat, 35% kcal carbohydrates, 20% kcal protein) for 32 weeks. Scale bar = 100 μm. Boxes indicate regions of higher magnification. (B) Blood ketone body levels at baseline and 6- and 24-hour post-fasting (SC, n = 7; HFD, n = 10). (C) Schematic of the hepatic ketogenic pathway with key metabolites and enzymes. (D) mRNA expression analysis of ketogenic enzymes in the liver, including Acat1 , Hmgcl , Hmgcs2 , and Bdh1 , in fed (SC, n = 6; HFD, n = 5) and 24-hour fast (SC, n = 7; HFD, n = 5). Hmgcs2 protein expression and quantification in (E) SC-fed healthy and (F) HFD-fed NAFLD mouse livers in fed and 24-hour fast (n = 3/group). Data are represented as mean ± SEM. Statistical analysis was performed by student's t -test and two-way or two-way repeated measures ANOVA. ∗ P ≤ 0.05; ∗∗ P ≤ 0.01; ∗∗∗ P ≤ 0.001 ∗∗∗∗ P ≤ 0.0001.

Article Snippet: Lipid droplets were visualized by staining with hematoxylin and eosin (H&E) or immunohistochemistry with anti-Perilipin 2 (Plin2) antibody (1:150, NB110-40877; Novus Biologicals) and the VECTASTAIN Elite ABC reagent (PK-6100; Vector Laboratories).

Techniques: Expressing, Immunohistochemical staining, Immunohistochemistry

Journal: Molecular Metabolism

Article Title: Hmgcs2-mediated ketogenesis modulates high-fat diet-induced hepatosteatosis

doi: 10.1016/j.molmet.2022.101494

Figure Lengend Snippet: Ketogenic deficiency through Hmgcs2 knockout results in fatty liver development in postnatal mice. (A) Liver Hmgcs2 protein expression in wild-type (WT), Hmgcs2 -heterozygous (HET) and knockout (KO) mice. (B) Schematic representing the postnatal stages of p0, p4, and p14 at which the mice were examined. (C) Hmgcs2 gene expression in the liver, (D) blood ketone levels, and (E) liver weights during postnatal development (p0: WT, n = 5; HET, n = 6–9; KO, n = 6; male and female combined/p4: WT, n = 7–9; HET, n = 6–11; KO, n = 4/p14: WT, n = 4–11; HET, n = 3–9; KO, n = 3–10). (F) Representative image of livers of p14 WT and KO mice. (G) H&E and (H) anti-Plin2 IHC stainings of p14 WT, HET and KO mouse liver sections. Scale bar = 100 μm. Boxes indicate regions of higher magnification. (I) Histological liver fat quantification and (J) NAFLD Activity Score (NAS) (WT, n = 6; HET, n = 5; KO, n = 7). (K) Liver triglyceride concentration of p14 WT, HET and KO mice (WT, n = 5; HET, n = 4; KO, n = 6). (L) Plin2 gene expression in the liver during postnatal development (p0: WT, n = 5; HET, n = 6; KO, n = 6; male and female combined/p4: WT, n = 7; HET, n = 6; KO, n = 4/p14: WT, n = 6; HET, n = 5; KO, n = 7). (M) Lipid accumulation markers ( Pparg , Fsp27 ), (N) lipid synthesis genes ( Srebp1c , Acc1 , Fasn ) and (O) lipid oxidation genes ( Ppara , Cpt1a , Mcad ) in p14 WT, HET and KO mouse livers (WT, n = 6; HET, n = 5; KO, n = 7). Data are represented as mean ± SEM. Statistical analysis was performed by one- or two-way ANOVA. ∗ P ≤ 0.05; ∗∗ P ≤ 0.01; ∗∗∗ P ≤ 0.001; ∗∗∗∗ P ≤ 0.0001. (Created with BioRender.com).

Article Snippet: Lipid droplets were visualized by staining with hematoxylin and eosin (H&E) or immunohistochemistry with anti-Perilipin 2 (Plin2) antibody (1:150, NB110-40877; Novus Biologicals) and the VECTASTAIN Elite ABC reagent (PK-6100; Vector Laboratories).

Techniques: Knock-Out, Expressing, Gene Expression, Activity Assay, Concentration Assay

Journal: Molecular Metabolism

Article Title: Hmgcs2-mediated ketogenesis modulates high-fat diet-induced hepatosteatosis

doi: 10.1016/j.molmet.2022.101494

Figure Lengend Snippet: Fatty liver disease in postnatal Hmgcs2 knockout mice is rescued by early weaning. (A) Schematic representation of the early weaning protocol in postnatal WT and Hmgcs2 -KO mice. Early-wean mice were separated from their mothers at p14 and transitioned from a high-fat breast milk to a standard laboratory chow diet. Suckling control mice remained on breast milk feeding until collection at postnatal day 21 (p21). (B) Hmgcs2 gene expression in the liver and (C) blood ketone levels of suckling and early-wean WT and Hmgcs2 -KO mice at p21. (D) Liver weights (suckling WT/KO, n = 4; early-wean WT/KO, n = 5). (E) Representative liver image of Hmgcs2 -KO suckling and early-wean mice at p21. (F) H&E and anti-Plin2 IHC staining of liver sections. Scale bar = 100 μm. Boxes indicate regions of higher magnification. (G) Histological liver fat quantification and (H) NAFLD activity score (NAS) (suckling WT, n = 5; suckling KO, n = 3; early-wean WT, n = 3; early-wean KO, n = 4; male and female mice combined). (I) Liver triglyceride concentrations of suckling and early-wean WT and Hmgcs2 -KO mice at p21 (suckling WT/KO, n = 4; early-wean WT/KO, n = 5). (J) Hepatic gene expression analysis of lipid accumulation markers ( Pparg , Fsp27 , and Plin2 ). Data are represented as mean ± SEM. Statistical analysis was performed by two-way ANOVA. ∗ P ≤ 0.05; ∗∗ P ≤ 0.01; ∗∗∗ P ≤ 0.001; ∗∗∗∗ P ≤ 0.0001. (Created with BioRender.com).

Article Snippet: Lipid droplets were visualized by staining with hematoxylin and eosin (H&E) or immunohistochemistry with anti-Perilipin 2 (Plin2) antibody (1:150, NB110-40877; Novus Biologicals) and the VECTASTAIN Elite ABC reagent (PK-6100; Vector Laboratories).

Techniques: Knock-Out, Control, Gene Expression, Immunohistochemistry, Activity Assay

Journal: Molecular Metabolism

Article Title: Hmgcs2-mediated ketogenesis modulates high-fat diet-induced hepatosteatosis

doi: 10.1016/j.molmet.2022.101494

Figure Lengend Snippet: Ketogenic insufficiency increased the susceptibility of HFD-induced NAFLD development and associated metabolic dysfunction. (A) Blood ketone levels in 8-week-old fed and 24-hour fasted WT and Hmgcs2- HET mice (pre-HFD) (WT, n = 6; HET, n = 7). (B) Schematic for assessment of NAFLD development in WT and Hmgcs2 -HET mice placed on 8-weeks of HFD. (C) Blood ketone levels in fed and 24-hour fasted post-HFD WT and Hmgcs2 -HET mice (WT, n = 7; HET, n = 9). (D) Liver weights (WT, n = 6; HET, n = 5). (E) Representative H&E and anti-Plin2 IHC staining of liver sections of WT and Hmgcs2 -HET mice. Scale bar = 100 μm. Boxes indicate regions of higher magnification. Data are represented as mean ± SEM. Statistical analysis was performed by student's t -test or two-way ANOVA. ∗∗∗ P ≤ 0.001; ∗∗∗∗ P ≤ 0.0001. (Created with BioRender.com).

Article Snippet: Lipid droplets were visualized by staining with hematoxylin and eosin (H&E) or immunohistochemistry with anti-Perilipin 2 (Plin2) antibody (1:150, NB110-40877; Novus Biologicals) and the VECTASTAIN Elite ABC reagent (PK-6100; Vector Laboratories).

Techniques: Immunohistochemistry

Journal: Molecular Metabolism

Article Title: Hmgcs2-mediated ketogenesis modulates high-fat diet-induced hepatosteatosis

doi: 10.1016/j.molmet.2022.101494

Figure Lengend Snippet: HMGCS2 overexpression ameliorates lipid accumulation in HepG2 cells. (A) Schematic representing timeline of in vitro experiment, starting with oleic acid treatment at 0-hours, adenovirus-mediated overexpression of GFP (Ad- GFP ) or HMGCS2 (Ad- HMGCS2 ) at 24-hours and cell collection at 48-hours. (B) RT-qPCR (n = 4/group) and (C) western blot quantification of HMGCS2 expression (n = 3/group) in HepG2 cells after Ad- HMGCS2 infection, compared to Ad- GFP . (D) Representative images of Oil-red-O staining and (E) its quantification of Ad- GFP or Ad- HMGCS2 treated HepG2 cells in the absence (−) and presence (+) of oleic acid (n = 3/group). (F) RT-qPCR of lipid synthesis ( SREBP1C , ACC1 , FASN ) and lipid accumulation ( FSP27 , PLIN2 ) genes in Ad- GFP and Ad- HMGCS2 treated HepG2 cells (n = 4/group). Data are represented as mean ± SEM. Statistical analysis was performed by student's t -test or two-way ANOVA. ∗ P ≤ 0.05; ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001; ∗∗∗∗ P ≤ 0.0001. (Created with BioRender.com).

Article Snippet: Lipid droplets were visualized by staining with hematoxylin and eosin (H&E) or immunohistochemistry with anti-Perilipin 2 (Plin2) antibody (1:150, NB110-40877; Novus Biologicals) and the VECTASTAIN Elite ABC reagent (PK-6100; Vector Laboratories).

Techniques: Over Expression, In Vitro, Quantitative RT-PCR, Western Blot, Expressing, Infection, Staining