Figure 5. Gluconeogenesis is impaired in hepatic Vps15 mutants.

(a) Relative mRNA expression levels of genes implicated in gluconeogenesis in the livers of 1-month-old random-fed Vps15^f/f and AlbCre⁺;Vps15^f/f mice. Data are means±s.e.m. (n=5–8, *P<0.05 versus Vps15^f/f, two-tailed, unpaired Student's t-test). (b) Immunoblot analysis of total protein liver extracts of random-fed 1-month-old Vps15^f/f and AlbCre⁺;Vps15^f/f using G6PC antibody. Immunoblot with anti-tubulin antibody served as loading control. (c) Intraperitoneal pyruvate tolerance test performed on overnight-starved 6-week-old Vps15^f/f and AlbCre⁺;Vps15^f/f mice (n=5–8, *P<0.05 versus Vps15^f/f, two-tailed, unpaired Student's t-test). The histogram of an average value of the area under the respective curve was calculated with GraphPadPrizm5 software (n=5–8, *P<0.05 versus Vps15^f/f, two-tailed, unpaired Student's t-test). (d) Immunoblot analyses of cell extracts of primary hepatocytes transduced with Adeno-shRNA SCR or Adeno-shRNA Vps15 vectors with indicated antibodies. Primary hepatocytes were transduced and cells were collected 48 h postinfection for further analysis. Immunoblot with anti-tubulin antibody served as a loading control. (e) Relative mRNA expression levels of gluconeogenic enzymes in primary hepatocytes transduced with Adeno-shRNA SCR or Adeno-shRNA Vps15 vectors. Cells were collected 48 h postinfection. Data are means±s.e.m. (n=3, *P<0.05 versus shRNA SCR infected, two-tailed, unpaired Student's t-test). (f) Glucose production by primary hepatocytes transduced with Adeno-shRNA SCR or Adeno-shRNA Vps15 vectors. Primary hepatocytes 48 h postinfection were incubated for 12 h in glucose-free DMEM containing lactate/pyruvate (10:1 mM). Glucose content in the media was assayed enzymatically and normalized to protein content. Data are means±s.e.m. (n=6, *P<0.05 versus shRNA SCR infected, two-tailed, unpaired Student's t-test).