Metabolic phenotype of NEMO^L-KO mice on NCD and HFD. (a) Body weights of WT mice on a NCD (open circles) and a HFD (open squares) were compared with NEMO^L-KO mice on NCD (filled circles) and HFD (filled squares). The weight of nine mice was determined weekly for each genotype. (b) The epigonadal fat pad weight of WT (white bars) and NEMO^L-KO (black bars) on NCD and on HFD was measured at week 19 (n = 9 per genotype). (c) Body compositions of WT (white bars) and NEMO^L-KO (black bars) on NCD and on HFD were determined by using a Brucker minespec (n = 7 per genotype). (d) Glucose tolerance tests of WT mice on NCD (open circles), on HFD (open squares) and of NEMO^L-KO mice on NCD (filled circles) and on HFD (filled squares) were performed at week 12 (n = 9 per genotype). (e) Insulin tolerance tests of mice with the indicated genotypes were performed at week 13 (n = 9 per genotype). (f) Serum insulin levels were determined by ELISA from sera of mice with the indicated genotypes at 8 and 16 weeks of age (n = 9 per genotype). (g) Fasted blood glucose was evaluated at week 13 (n = 9 per genotype). (h) Relative expression of gluconeogenic enzymes PEPCK and G6P were determined by real-time PCR from liver samples of the indicated genotypes (n = 8 per genotype) using TaqMan Assay. (i) Representative insulin signaling of mice with the indicated genotypes by using Western blot analysis with the indicated antibodies. Mice were injected into the vena cava with 125 μl of 40 units/ml insulin upon anesthetization. Values are mean ± SEM. *, P ≤ 0.05; **, P ≤ 0.001 vs. control.

Massive macrophage infiltration into livers of NEMO^L-KO mice leads to up-regulation of proinflammatory cytokines, induction of cell death and compensatory proliferation. (a) Immunohistochemistry of liver cryostat sections of control and NEMO^L-KO mice using F4/80 antibody to detect infiltrating macrophages. (b) Relative expression of MCP-1 and TNF-α in livers of control and NEMO^L-KO mice on NCD and HFD was determined by real-time PCR using TaqMan Assay (n = 8 per genotype). (c) Evaluation of IL-6 levels was performed by ELISA from sera of control and NEMO^L-KO mice on NCD and HFD at 16 weeks of age. (d) Representative Western blots of liver protein lysates from control and NEMO^L-KO mice on NCD and HFD using the indicated antibodies (n = 3 per genotype). (e) Quantitation of P-STAT-3 intensities from Western blots shown in d by using Quantity one software. (f) Determination of apoptosis by TUNEL staining of livers of control and NEMO^L-KO mice was expressed as percentage of total cells. TUNEL-stained liver sections were counterstained with DAPI and from five exemplary sections (see SI Fig. 7) for each value DAPI as well as TUNEL-positive cells were counted (n = 5 per genotype). Values are mean ± SEM. *, P ≤ 0.05; **, P ≤ 0.001 vs. control.

Macrovesicular steatosis in livers of NEMO^L-KO mice. (a) Representative Sudan staining from liver sections of control and NEMO^L-KO mice on NCD and HFD at 5 months of age. Sections were counterstained with hematoxylin. (b) Percentage of macrovesicular lipid accumulations in lipid containing hepatocytes. (c) Relative expression of the indicated genes from the different genotypes was performed by real-time PCR by using TaqMan Assay (n = 8 per genotype). Values are mean ± SEM. *, P ≤ 0.05; **, P ≤ 0.001 vs. control. (d) Triglyceride and (e) cholesterol levels from sera of control and NEMO^L-KO mice on NCD and HFD at week 16 were determined by a diagnostic laboratory using standard techniques (n = 16 per genotype).

HFD drastically elevates spontaneous tumorigenesis in livers of NEMO^L-KO mice. (a) Exemplary demonstration of ex situ isolated livers of control and NEMO^L-KO mice on NCD and HFD. (b) Macroscopic quantitation of large (>2-mm) and small (≤2-mm) foci of NEMO^L-KO mice on NCD and HFD (n = 5 per genotype) at week 19. (c) Representative paraffin sections of control and tumorigenic liver tissue from NEMO^L-KO mice on NCD and HFD. (d) Determination of proliferating cells in livers of control and NEMO^L-KO mice either on NCD or HFD was performed by immunohistochemistry using Ki-67 antibody. (e) Microscopic quantitation of foci in livers of NEMO^L-KO mice on a NCD and a HFD (n = 5 per genotype). A value of 1 expresses one to three foci/lobule, whereas a value of 2 represents more than three foci/lobule. Values are mean ± SEM. *, P ≤ 0.05; **, P ≤ 0.001 vs. control.