(A) A 2-hr hyperinsulinemic-euglycemic clamp was performed in conscious mice following exposure to HFD for 12 weeks. Glucose infusion rates, whole body glucose turnover, and hepatic glucose production were measured at basal levels (prior to insulin clamp) and during the insulin-stimulated state. Tissues were taken at the end of the clamp experiments to assess insulin-stimulated glucose uptake in skeletal muscle (gastrocnemius), heart, and white and brown adipose tissues. Data are expressed as mean ± S.E. of 5 to 6 animals per group. *p < 0.05 versus WT. (B) Mice were fasted overnight and treated with insulin (5 U/kg body weight) for 10 min by intraperitoneal injection. Liver, WAT, and muscle tissues were isolated and examined by western blot analysis using antibodies to phospho-AKT, AKT, and Actin.

(A) Experimental design for the generation of hematopoietic and non-hematopoietic PKCζ^-/- mice. (B) Quantification by flow cytometry of total white blood cell reconstitution after transplantation. PKCζ^-/- cells express the CD45.2 (Ly5.1) marker, whereas WT cells express the CD45.1 (Ly5.2) marker. (C) PCR genotyping of white adipose tissue (WAT), muscle, and bone marrow-derived macrophages (BMDM) obtained from chimeras 23 weeks after transplantation. (D) Body-weight changes during experimental period. (E) Glucose tolerance test. (F) Insulin tolerance test. (G) Gross morphology of epididymal adipose tissues. (H) Sections of epididymal adipose tissues stained with hematoxylin and eosin (upper panel) and F4/80 (lower panel). The expression of CD68 (I) and F4/80 (J) were measured by QRT-PCR. (K) Gross liver morphology. (L) Liver sections were stained with hematoxylin and eosin. (M and N) The amount of plasma IL-6 was measured by ELISA. Mice were fasted overnight before collecting blood. (M) Chimeras fed with HFD for 20 weeks. (N) WT and PKCζ^-/- mice fed a RD or HFD for 12 weeks. Magnifications shown are 10X. Scale bars, 200 μm. Data are mean ± S.E. of 5 to 7 animals per group. *p < 0.05 versus WT+WT-BM or WT.

(A) Stromal-vascular cells were differentiated to adipocytes and treated with IL-4 (10 ng/ml) or vehicle control for 48 hrs and subsequently stimulated with LPS 100 ng/ml for an additional 8 hrs. Relative expression of IL-6 was quantified by QRT-PCR. (B) The phosphorylation of Stat-6 after IL-4 treatment (10 ng/ml for 30 min) was measured in adipocyte-differentiated stromal-vascular cells by western blot analysis. (C-F) IL-6 secretion was measured by ELISA. (C) Epididymal adipose tissue (EP WAT). (D) Retroperitoneal adipose tissue (RP WAT). Adipose tissues were placed in medium for 72 hrs. IL-6 was not detected in medium from IL-6^-/- or DKO RP WAT (ND, no detection). (E) IL-6 secretion was measured during 3T3-L1 adipocyte-differentiation after lentiviral knockdown of PKCζ (shPKCζi) or in negative control cells (shNT). Cells were treated with differentiation cocktail for 3 days. (F) IL-6 secretion was measured in fully adipocyte-differentiated stromal-vascular cells after stimulation with different free fatty acids for 16 hrs. Ctrl, control; MA, myristic acid (50 mM); PA, palmitic acid (50 mM). (G) Adipocyte-differentiated stromal-vascular cells were stained with Oil-red O at day 8 after stimulation with differentiation cocktail. (H) PKCζ protein expression was measured by western blot analysis of adipose tissue from RD- or HFD-fed WT mice. Actin was used as a loading control. (I) The expression of PKCζ protein was measured in adipose tissue from age-matched WT or ob/ob mice. Tubulin was used as a loading control. Scale bars, 200 μm. *p < 0.05 versus WT.

(A) Genotypic analysis of mice by PCR. (B) PKCζ protein expression in different tissues from wild-type (WT) and PKCζ^-/- mice. (C) Glucose tolerance test (GTT). (D) Insulin tolerance test (ITT). (E) Glucose-stimulated insulin secretion was measured in WT and PKCζ^-/- mice fed a high-fat diet (HFD) for 12 weeks. (F) Resting blood insulin was examined in WT and PKCζ^-/- mice fed a regular diet (RD) or HFD as above. (G) Fasting blood glucose levels. (H-J) Hepatic gluconeogenic gene expression. (H) PGC1α, Peroxisome proliferator-activated receptor gamma coactivator-1 alpha. (I) G6Pase, glucose 6 phosphatase. (J) PEPCK, Phosphoenolpyruvate carboxykinase. Body weights (H) and grams of total fat (I) were measured in mice fed a RD or HFD for 12 weeks. Data are mean ± S.E. of 6 to 7 animals per group. *p < 0.05 versus WT.

(A) Epididymal adipose tissues and (B) interscapular adipose tissues were isolated from WT and PKCζ^-/- mice fed a RD or HFD for 12 weeks. (C) Epididymal adipose tissue sections were stained with hematoxylin and eosin. (D) Adipocyte area was evaluated in around 500 cells in sections of epididymal adipose tissue from 3 different animals per group. (E) Sections of epididymal adipose tissue used for measurements in panel (D) immunostained against F4/80. (F and G) The expression of the CD68 and F4/80 was examined by QRT-PCR in epididymal adipose tissue. (H) Gross liver morphology. (I) Liver weights were measured in mice fed a RD or HFD for 12 weeks. (J) Liver sections stained with hematoxylin and eosin. (K) Liver triglyceride contents measured by using an enzymatic method (Randox)s. Scale bars, 200 μm. Data are mean ± S.E. of 6 to 7 animals per group. *p < 0.05 versus WT.

WT, IL-6^-/-, PKCζ^-/-, and PKCζ/IL-6 double knockout (DKO) mice were fed a HFD for 12 weeks from 7 weeks of age. (A) Changes in body weight over 12 weeks of HFD treatment. (B) Fat mass (percentage of total body weight) after 12 weeks of HFD treatment. (C) Glucose tolerance test (GTT). (D) Insulin tolerance test (ITT). In this ITT, 1.25 U insulin/kg body weight was injected. Sections of epididymal adipose tissues were stained with hematoxylin and eosin (E) or immunostained to detect F4/80 (F). Expression of CD68 (G) and F4/80 (H) was measured by QRT-PCR. Liver sections were stained with hematoxylin and eosin (I). Scale bars, 200 μm. Data are mean ± S.E. of 5 to 7 animals per group. *p < 0.05 versus WT.