1. The Protein Kinase IKKɛ Regulates Energy Balance in Obese Mice
Shian-Huey Chiang, Merlijn Bazuine, Carey N. Lumeng, Lynn M. Geletka, Jonathan Mowers, Nicole M. White, Jing-Tyan Ma, Jie Zhou, Nathan Qi, Dan Westcott, Jennifer B. Delproposto, Timothy S. Blackwell, Fiona E. Yull, Alan R. Saltiel 
Cell 
Volume 138, Issue 5, Pages (September 2009)
DOI: /j.cell
Copyright © 2009 Elsevier Inc. Terms and Conditions

2. Figure 1
HFD Increases NF-κB Activity in Adipose Tissue Measured by In Vivo Bioluminescence in Live Mice
(A) Male HLL mice on normal diet (ND) and HFD were assessed for bioluminescence after injection of luciferin. Luminescence was collected from a gate limited to the abdominal cavity is presented for ND and HFD HLL mice. (n = 7 per group). ∗p value < 0.05.
(B) Tissues from HLL mice were dissected and assessed ex vivo for luminescence. Epididymal white adipose tissue (Epi WAT).
(C) Quantitation of absolute tissue luminescence from HLL mice. n = 7 per group. Data was collected serially after dissection to ensure plateau of luminescent signal.
(D) Immunohistochemical localization of luciferase expression in epididymal fat pads from HLL mouse fed with normal and high-fat diet.
(E) Epididymal fat pads from ND or HFD fed male mice were analyzed for p65/RelA expression by immunofluorescence showing maximal signal concentration of p65/RelA in ATM clusters and localization of p65 in ATM nuclei (TOPRO3 as nuclear marker). The scale bar represents 20 μm.
Cell  , DOI: ( /j.cell )
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3. Figure 2
HFD Increases IKKɛ Expression in White Adipose Tissue and Liver
(A) qPCR analysis on the expression of genes encoding IKK family members in liver and white adipose tissue. White bars, wild-type mice, normal diet (ND) (n = 6); gray bar, wild-type mice, high-fat diet (HFD) for 4 months (n = 6). All data are presented as the average ± SEM normalized to Rplp0 expression. Average of ND value was set as 1.
(B) qPCR analysis on the expression of genes encoding IKK family members in isolated adipocytes and stromal vascular fraction. White bars, ND (n = 6); gray bar, HFD for 4 months (n = 6).
(C) (Top) confocal merged images from epididymal fat pads from ND and HFD-fed mice co-stained with anti-IKKɛ (green), anti-MGL1 (red) and anti-Caveolin1 (blue) antibodies. Representative image shown and similar results from 3-4 independent mice. (Bottom) confocal images from epididymal fat pads from HFD-fed wild-type mice immunostained with anti-IKKɛ antibody (green), anti-F4/80 antibody (red) or merged image with anti-caveolin 1 antibody (blue). The scale bar represents 50 μm.
(D) Lysates from liver and white adipose tissue (WAT) of wild-type (WT) and IKKɛ knockout mice (IKKɛ KO) fed with ND or HFD were immunoprecipitated with antibody against IKKɛ as indicated. The expression level of IKKɛ was determined by immunoblotting with same antibody against IKKɛ.
(E) Lysates from liver and WAT of WT and IKKɛ KO fed with ND or HFD were immunoprecipitated (IP) with antibody against IKKɛ and assayed for kinase activity against myelin basic protein (MBP) as substrate. The expression level of IKKɛ in IP was determined by immunoblotting with same antibody against IKKɛ. Lysates for IP were immunoblotted with antibodies against Rab5B and Caveolin 1 as a loading control.
Cell  , DOI: ( /j.cell )
Copyright © 2009 Elsevier Inc. Terms and Conditions

4. Figure 3
IKKɛ KO Mice Are Protected from Diet-Induced Weight Gain by Increasing Energy Expenditure
(A) Representative images of mice from WT and IKKɛ KO mice fed with ND or HFD.
(B) Representative confocal image of caveolin-stained epididymal adipose tissue from WT and KO mice fed with HFD. The scale bar represents 100 μm.
(C) Whisker plot of adipocyte area from evaluation of < 500 adipocytes from 3–4 independent mice. ∗p < comparing mean adipocyte area.
(D) Adipocyte numbers in fat pads of WT (gray bar) and IKKɛ KO mice (black bar) fed with HFD. n = 5 mice per genotype. ∗, p value <
(E) Food intake was measured for WT (gray bar) and KO (black bar) mice fed with ND or HFD as indicated. n = 8 mice per group. ∗, p value < 0.05.
(F) Oxygen consumption (VO2) was measured for WT (orange) and KO (green) mice fed with ND (top) or WT (red) and KO (blue) mice fed with HFD (bottom) for three days as indicated. n = 8 mice per group.
(G) Respiratory quotient (RQ) was measured for WT (orange) and KO (green) mice fed with ND (top) or WT (red) and KO (blue) mice fed with HFD (bottom) for three days as indicated. n = 8 mice per group.
(H) (Top) qPCR analysis on the expression of genes encoding UCP1 in WAT. Gray bars, wild-type mice (n = 6); black bar, IKKɛ KO mice (n = 6) fed with ND or HFD as indicated. All data are presented as the average ± SEM normalized to Rplp0 expression. ∗, p value < Average of WT fed with ND value was set as 1. (Bottom) Protein expression of UCP1 in WAT, measured by immunoblotting with WAT lysates from WT and IKKɛ KO mice (5 mice in each group) fed with HFD as indicated. Rab5 was used as internal loading control.
(I) Rectal temperature measured for WT and KO mice fed with ND (3 months old) or HFD (5 months old with diet for 2 months). n = 10 per group. ∗, p value < 0.05; ∗∗, p value < 0.01.
Cell  , DOI: ( /j.cell )
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5. Figure 4 IKKɛ KO Mice Display Improved Glucose and Lipid Homeostasis
(A) Blood glucose and serum insulin levels measured for 18 hr fasting WT (gray bar) and KO (black bar) mice fed with ND or HFD as indicated. n = 12 mice per group.
(B) Serum NEFA, triglyceride and total cholesterol levels measured for 18 hr-fasting WT (gray bar) and KO (black bar) mice fed with ND or HFD as indicated. n = 12 mice per group.
(C) Glucose tolerance test (GTT) measured for 12 hr-fasting WT (gray) and KO (black) mice fed with ND (left panel) or HFD (right panel). n = 12 mice per group.
(D) Serum insulin levels measured for mice during GTT shown in (C) at time points 0, 30, 60 and 180min after injection.
(E) Insulin tolerance test (ITT) measured for 3 hr fasting WT (gray) and KO (black) mice fed with ND or HFD. n = 12 mice per group.
(F) Pyruvate tolerance test (PTT) measured for 12 hr fasting WT (gray) and KO (black) mice fed with HFD. n = 12 mice per group. All data are presented as the average ± SEM (∗∗, p value < 0.01).
Cell  , DOI: ( /j.cell )
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6. Figure 5
IKKɛ KO Preserves Insulin Signaling and Insulin Sensitivity in Liver and Adipose Cells in Mice on HFD
(A-C) Mice fasted for 18 hr were IP-injected with insulin (5 mU/g) or saline. Lysates from liver (A), WAT (B) and gastrocnemius (C) of WT, or IKKɛ KO mice fed with HFD were immunoblotted with indicated antibodies.
(D and E) qPCR analysis on the expression of genes encoding PDK4 (D) and glucokinase (E) in liver of WT and IKKɛ KO mice fed with ND or HFD as indicated. Gray bars, wild-type mice (n = 6); black bar, IKKɛ KO mice (n = 6).
(F and G) qPCR analysis on the expression of genes encoding adiponectin (F) and PPARγ (G, Top) in WAT of WT and IKKɛ KO mice fed with ND or HFD as indicated. Gray bars, wild-type mice (n = 6); black bar, IKKɛ KO mice (n = 6). (G, Bottom) protein expression of PPARγ in WAT, measured by immunoblotting with WAT lysates from WT and IKKɛ KO mice (5 mice in each group) fed with HFD as indicated. Rab5 was used as internal loading control.
(H) (Left) qPCR analysis on the expression of genes encoding the PPARγ targets CD36, CAP, GLUT4 in WAT of WT and IKKɛ KO mice fed with ND or HFD as indicated. Gray bars, wild-type mice (n = 6); black bar, IKKɛ KO mice (n = 6). (Right) protein expression of CD36, CAP and GLUT4 in WAT, measured by immunoblotting with WAT lysates from WT (duplicate mice in each group) and IKKɛ KO mice (triplicate mice in each group) fed with ND or HFD as indicated. Rab5 was used as internal loading control.
(I) Ex vivo insulin-stimulated glucose incorporation into lipid in adipocytes isolated from WT and KO mice fed with ND or HFD as indicated. Cells were untreated (white bar) or treated with insulin for 60 min (shaded bar). n = 3 mice per condition. (∗∗, p value < 0.01).
(J) Insulin-stimulated glucose uptake in 3T3-L1 adipocytes. Differentiated adipocytes were electroporated with vector control (white bar), IKKɛ WT (gray bar) or IKKɛ kinase dead (K38A) (black bar) mutant expression constructs. Cells were untreated (basal) or treated with insulin for 30min (Insulin). 14C-2DG uptake in cells was normalized with total amount of protein. n = 3 for each condition. All data are presented as the average ± SEM.
Cell  , DOI: ( /j.cell )
Copyright © 2009 Elsevier Inc. Terms and Conditions

7. Figure 6
IKKɛ KO Mice Are Protected from Diet-Induced Hepatic Steatosis
(A) Liver weight normalized with body weight was measured from WT (gray bar) and IKKɛ KO (black bar) mice fed with ND or HFD as indicated. n = 8 for each group. (∗, p value < 0.05).
(B) Representative images of liver from WT and KO mice fed with ND or HFD as indicated.
(C) Liver triglyceride content normalized with liver weight was measured from WT (gray bar) and IKKɛ KO (black bar) mice fed with ND or HFD in fed or fasted condition as indicated. n = 8 for each group (∗, p value < 0.05; ∗∗, p value < 0.01).
(D) Representative images of hematoxylin and eosin-stained section of liver from fasting WT or KO mice fed with HFD for 2 months. Arrows indicate central veins.
(E) (Top) qPCR analysis on the expression of the gene encoding Lipin1 in liver of WT and IKKɛ KO mice fed with ND or HFD as indicated. Gray bars, wild-type mice (n = 6); black bar, IKKɛ KO mice (n = 6). (∗, p value < 0.05). (Bottom) protein expression of lipin1 in liver, measured by immunoblotting with liver lysates from WT (duplicate mice in each group) and IKKɛ KO mice (triplicate mice in each group) fed with ND or HFD as indicated. Rab5 was used as an internal loading control.
(F) qPCR analysis on the expression of genes encoding CD36, FABP4, PPARγ in liver of WT and IKKɛ KO mice fed with ND or HFD as indicated. Gray bars, wild-type mice (n = 6); black bar, IKKɛ KO mice (n = 6). (∗, p value < 0.05; ∗∗, p value < 0.01).
(G) (Top) Immunoblotting with an anti-FLAG antibody in H2.35 hepatoma cells. (Bottom) qPCR analysis on the expression of the indicated genes. Gene expression was measured from cells transfected with vector control (white bar), IKKɛ WT (gray bar) or IKKɛ kinase dead (K38A) (black bar) mutant expression constructs. (∗, p value < 0.05; ∗∗, p value < 0.01).
Cell  , DOI: ( /j.cell )
Copyright © 2009 Elsevier Inc. Terms and Conditions

8. Figure 7 Obesity-Induced Inflammation Is Attenuated in IKKɛ KO Mice
(A) Serum proinflammatory cytokines MCP-1, TNF-α and Rantes secretion were measured in WT (gray bar) and IKKɛ KO mice (black bar) fed with ND or HFD as indicated. n = 8. (∗∗, p value < 0.01).
(B) Representative images from epididymal fat pads of WT and KO mice fed with HFD co-stained with F4/80 (green), Isolectin (red) and caveolin 1 (blue).
(C) Quantitation of F4/80+ crown-like structures. Confocal images were used to quantitate the percentage of crown-like structures. 3-5 low power fields analyzed for 3-4 mice per genotype (>1000 adipocyte examined per genotype, ∗p-value < 0.001).
(D) qPCR analysis on the expression of genes encoding TNF-α, Rantes, MIP-1α, IP 10 and MCP-1 in WAT of WT and IKKɛ KO mice fed with ND or HFD as indicated. Gray bars, wild-type mice (n = 6); black bar, IKKɛ KO mice (n = 6). (∗, p value < 0.05; ∗∗, p value < 0.01).
(E) qPCR analysis on the expression of genes encoding TNF-α, MCP 1, MIP 1α, IP 10 and Rantes in liver of WT and IKKɛ KO mice fed with ND or HFD as indicated. Gray bars, wild-type mice (n = 6); black bar, IKKɛ KO mice (n = 6). (∗, p value < 0.05; ∗∗, p value < 0.01).
(F) qPCR analysis on the expression of gene encoding iNOS in liver of WT and IKKɛ KO mice fed with ND or HFD as indicated. Gray bars, wild-type mice (n = 6); black bar, IKKɛ KO mice (n = 6). (∗∗, p value < 0.01).
(G) Protein level of phospho-JNK, JNK, IκB were measured by immunoblotting with lysates from liver, gastrocnemius and WAT of WT (duplicate mice in each group) and IKKɛ KO mice (triplicate mice in each group) fed with ND or HFD as indicated. Rab5 and caveolin 1 were used as internal loading controls.
All data are presented as the average ± SEM.
Cell  , DOI: ( /j.cell )
Copyright © 2009 Elsevier Inc. Terms and Conditions