Effect of HF diet on proteins related to hepatic lipid metabolism and on hepatic glutathione (GSH) levels in male Cc1^+/+ and Cc1^−/− mice. Male wild-type Cc1^+/+ and Cc1^−/− mice were fed a regular chow (RD) or a HF diet for 3 months, starting at 3 months of age. A) Livers were then removed and analyzed by immunoblotting (iB) with α-FAS and α-NPC1, followed by reimmunoblotting (reiB) with α-actin and α-RPS3 antibodies, respectively, to normalize for the amount of proteins loaded, as described in Materials and Methods. The gel represents at least 3 mice per feeding group. B) Serum (10 μg) was analyzed, as above, by immunoblotting using α-ApoB antibody to detect ApoB100 and ApoB48 proteins. Although 2 mice are included in the figure, these experiments were performed on at least 5 mice per feeding group. C) Effect on hepatic GSH levels. N > 7 mice per feeding group. Values are mean ± SEM. ^aP < 0.005 HF versus RD. ^bP < 0.05 Cc1^−/− versus Cc1^+/+ in the same feeding group.
Abbreviations: Cc1^−/−, global Ceacaml null mouse; Cc1^+/+, wild-type mouse from the same genetic background as Cc1^−/− mice; RD, regular diet; HF, high-fat diet; FAS, fatty acid synthase; NPC1, Niemann Pick type C1; ApoB, apolipoprotein B; GSH, glutathione; iB, immunoblotting; reiB, reimmunoblotting.

Effect of HF diet and genotype on the presence of liver inflammatory infiltrate. a) Representative liver sections from n > 4 mice stained with H&E. HF-fed Cc1^−/− mouse livers (right panel) reveal increased perivascular and lobular inflammatory cell infiltrate relative to livers from HF-fed Cc1^+/+ mice (left panel) that had fewer and mostly portal inflammatory cell foci. b) Liver lysates were analyzed by sequential immunoblotting (iB) with α-phospho-NF-κB (upper gel), followed by α-NF-κB (lower gel) antibodies as an index of inflammation. The gel represents at least 3 mice from each feeding group.
Abbreviations: Cc1^−/−, global Ceacaml null mouse; Cc1^+/+, wild-type mouse from the same genetic background as Cc1^−/− mice; RD, regular diet; HF, high-fat diet; H&E staining, hematoxylin-eosin staining; iB, immunoblotting; reiB, reimmunoblotting.

Effect of HF diet and genotype on liver cell apoptosis. a) Liver lysates were analyzed by sequential immunoblotting (IB) with antibodies against caspase 3 (upper gel), followed by α-actin (lower gel) to detect cleaved caspase 3 (17 kdaltons) as an index of apoptosis. The gel represents at least three mice from each feeding group. b) Liver sections from n > 4 mice per feeding group were analyzed by TUNEL staining for apoptotic cells, as described in Materials and Methods. Representative sections are shown. Panel 1: HF-fed Cc1^+/+; Panel 2, HF-Fed Cc1^−/−, Apoptotic cells are observed only in Cc1^−/− livers. Panels 1-c and 2-c are negative controls without TDT enzyme following digestion with proteinase K to account for nonspecific binding of enzyme conjugate. Sections from mice on the regular diet are not shown because there was little evidence of apoptosis in either genotype.
Abbreviations: Cc1^−/−, global Ceacaml null mouse; Cc1^+/+, wild-type mouse from the same genetic background as Cc1^−/− mice; RD, regular diet; HF, high-fat diet; 1B, immunoblotting; re1B, reimmunoblotting.

Effect of HF diet on liver histology in male wild-type Cc1^+/+ and Cc1^−/− mice. Liver histology was assessed in H&E stained sections (n > 4 mice per feeding group), as described in Materials and Methods. Panel 1: Cc1^+/+, regular diet; Panel 2: Cc1^+/+ HF diet; Panel 3: Cc1^−/−, regular diet; Panel 4: Cc1^−/−, diet. Representative sections are shown. in HF-fed Cc1^+/+ (panel 2), the lipid infiltration alternates with normal liver parenchyma and is predominantly microvesicular. in contrast, in HF-fed Cc1^−/− (panel 4), the lipid infiltration appears more diffuse and predominantly macrovesicular.
Abbreviations: Cc1^−/−, global Ceacaml null mouse; Cc1^+/+, wild-type mouse from the same genetic background as Cc1^−/− mice; RD, regular diet; HF, high-fat diet; H&E, hematoxylin-eosin.

Effect of HF diet and genotype on liver fibrosis. Liver sections from n > 4 mice per feeding group were stained with Sirius red. Panel 2: RD-fed Cc1^−/− mice show mild pericellular fibrosis. Panel 4: HF-fed Cc1^−/− mice, show extensive pericellular and perivascular fibrosis.
Abbreviations: Cc1^−/−, global Ceacaml null mouse; Cc1^+/+, wild-type mouse from the same genetic background as Cc1^−/− mice; RD, regular diet; HF, high-fat diet.