Fig 1.
Hepatocyte-specific deletion of UBXD8 in mice.
(A) PCR genotyping. The 187-bp and 245-bp products were obtained from wild-type and Ubxd8flox/flox mice, respectively. (B) Western blot showing liver-specific depletion of UBXD8 in the UBXD8-LKO mouse. Tissue lysates were prepared from control (Cre-/-, Ubxd8flox/flox) and UBXD8-LKO (Cre+/-, Ubxd8flox/flox) mice, and an equal amount (30 μg) of protein was loaded for each lane in SDS-PAGE. (C) Western blot showing depletion of UBXD8 in hepatocytes isolated from the UBXD8-LKO mouse.
Fig 2.
Body weight, liver weight, and blood chemistry results of mice fed a normal diet.
Control and UBXD8-LKO mice (30 wk old) were fed a normal diet for 26 wk. 15 females (A) and 15 males (B) were examined for each group. Center lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range (IQR) from the 25th and 75th percentiles; and outliers are represented by dots. The notches are defined as ± 1.58*IQR/sqrt(n) and represent the 95% confidence interval for each median. Indices marked with an asterisk (*) differed significantly between the two groups (P < 0.05; non-paired Student's t test). Numerical data (means ± SEM) and data on additional indices are shown in Table 1. BW: body weight; LW: liver weight; Glu: glucose; FFA: free fatty acid; T-Cho: total cholesterol.
Table 1.
Body weight, liver weight, and blood chemistry results of mice fed a normal diet.
Fig 3.
Body weight, liver weight, and blood chemistry results of mice fed a high-fat diet.
Control and UBXD8-LKO mice (30 wk old) fed a high-fat diet for 26 wk. 15 females (A) and 15 males (B) were examined for each group. Indices marked with an asterisk (*) differed significantly between the two groups (P < 0.05; non-paired Student's t test). Box plots were prepared in the same manner as Fig 2. Numerical data (means ± SEM) and data on additional indices are shown in Table 2. BW: body weight; LW: liver weight; Glu: glucose; FFA: free fatty acid; T-Cho: total cholesterol.
Table 2.
Body weight, liver weight, and blood chemistry results of mice fed a high-fat diet.
Fig 4.
High-fat diet caused periportal steatosis in UBXD8-LKO mice.
(A) Histology of liver sections obtained from control and UBXD8-LKO mice (30 wk old) fed a high-fat diet for 26 wk. The perivenular area (zone 3) of both groups showed steatosis of the predominantly microvesicular type, whereas the periportal area (zone 1) showed macrovesicular steatosis only in UBXD8-LKO mice. CV: central vein, PV: portal vein. (B) Analysis of lipids extracted from livers of control and UBXD8-LKO mice fed a high-fat diet for 26 wk. TG, total cholesterol, and free cholesterol did not exhibit significant differences between the two groups (n = 5; means ± SEM).
Table 3.
Histological evaluation of liver at 30 wk of age.
Fig 5.
Analysis of serum lipoproteins.
(A) Lipoprotein profile obtained by gel filtration–HPLC of mouse serum at 30 wk old. A representative result on TG (blue) and total cholesterol (red) is shown for sera of normal and UBXD8-LKO male mice fed a normal (A) or a high-fat diet (B). Sera from female mice gave similar results. Sera for the UBXD8-LKO group fed a high-fat diet were taken from mice showing periportal steatosis. Lower panels show a magnified view of the VLDL portion. FG: free glycerol. (B) VLDL-TG was lower in UBXD8-LKO mice than in normal mice, even when they were fed a normal diet. The difference between the control and UBXD8-LKO mouse became significantly larger when mice were fed a high-fat diet. P values were obtained by non-paired Student's t test (n = 3; means ± SEM). (C) The TG-to-cholesterol ratio in VLDL tended to be lower in the UBXD8-LKO than in the control mouse. P values were obtained by non-paired Student's t test (n = 3; means ± SEM).
Fig 6.
TG secretion was lower in UBXD8-LKO mice than in controls.
TG secretion estimated by the Triton WR-1339 injection method. Control and UBXD8-LKO mice, both male and female, were fed a normal diet until 30–33 wk of age. Serum TG levels were measured before and 1 and 2 h after the Triton WR-1339 injection. P values were obtained by non-paired Student's t test (n = 4; means ± SEM).
Fig 7.
(A) Cultured primary hepatocytes obtained from the UBXD8-LKO mice secreted lower levels of ApoB than cells obtained from the control mice. Hepatocytes obtained from female mice fed a normal diet until 12 wk of age were cultured, and ApoB and albumin secreted into the culture medium were examined by densitometry of Western-blot signals. The albumin signal was used for normalization. The amount of secreted ApoB was lower in hepatocytes obtained from UBXD8-LKO mice than in those obtained from control mice, whether the cells were cultured in the normal medium (DMEM + 10% FBS) (left) or in the medium supplemented with 0.4 mmol/l OA (right). P values were obtained by non-paired Student's t test (n = 3; means ± SEM). (B) HepG2 cells transfected with control or UBXD8 siRNA were pulse-labeled with 35S-methionine and treated with 0.4 mmol/l oleic acid. ApoB immunoprecipitated from the medium at 1 and 3 h was subjected to SDS-PAGE and quantitated by radiography. ApoB secreted in the medium is shown as the ratio relative to total cellular ApoB immediately after pulse-labeling. (C) Primary hepatocytes obtained from UBXD8-LKO mice showed ApoB-crescent structures. The ApoB-crescent was observed as a crescent-shaped ApoB labeling (red) adjacent to LDs (green). Nuclei were stained with Hoechst dye (blue). The ApoB-crescent was present only in cells derived from UBXD8-LKO mice, and not in those obtained from control mice. Cells were cultured in the presence of 1.2 mmol/l OA.
Fig 8.
Diagram of ApoB lipidation and VLDL maturation in normal and UBXD8-deficient hepatocytes.
In normal hepatocytes, lipidated ApoB destined for proteasomal degradation is translocated to the cytoplasmic side and subjected to ubiquitination by a UBXD8-dependent mechanism. In the absence of UBXD8, lipidated ApoB accumulates at the ER–LD interface and disturbs the normal ApoB lipidation process. This probably results in a decrease in VLDL-TG secretion in UBXD8-null hepatocytes.