Reduced adiposity in Smad3-KO mice. A–E: Mean body weight (A), overall mouth-to-anus body length (B), and relative weight of subcutaneous (C), epididymal (D), and visceral (E) white adipose fat pads of 8-week-old WT and Smad3-KO mice. Values represent percentage of body weight; n= 8/group. F: Body fat content and lean mass composition analysis of WT and KO mice; n= 8/group. G: Representative hematoxylin–eosin-stained paraffin-embedded section of WT and KO epididymal WAT. Scale bars, 100 μm. H: Mean cross-sectional area of WT and KO adipocytes (n= 2,000/group). I: Mean adipocyte number in WT and KO epididymal WAT. J: Flow cytometry analysis of adipocytes (Nile Red positive) and stromal/vascular cells (Hoechst positive) in WT and KO epididymal WAT. WAT was subjected to collagenase digestion. The adipocyte layer was gently recovered and stained for 5 min with 10 μL of Nile Red staining solution (Molecular Probes, Eugene, OR). Cells positive for Nile Red were counted using LSR II Flow Cytometer System (Becton Dickinson, Franklin Lakes, NJ). Data are represented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. (A high-quality color representation of this figure is available in the online issue.)

Smad3-KO mice develop hypoglycemia and insulin hypersensitivity. A–E: Mean plasma triglyceride (A), FFA (B), glycerol (C), blood glucose (D), and blood insulin (E) in WT and KO mice. Blood glucose and insulin levels were measured at 6-h fasted and fed states. Blood glucose levels were measured using an Accu-Chek Advantage glucometer with glucose measurement strips (Roche Diagnostics, Indianapolis, IN). Serum insulin was measured by ELISA (Millipore, Billerica, MA). Serum total cholesterol, triglyceride, glycerol, and FFA concentrations were determined by quantitative enzymatic assays using l-type CHO-H (Wako Pure Chemical Industries, Osaka, Japan), serum triglyceride determination kit (Sigma-Aldrich, St. Louis, MO), free glycerol determination kit (Sigma-Aldrich), and serum/plasma FA and glycerol kit (Zen-Bio, Research Triangle Park, NC), respectively. F: Changes in blood glucose concentration at the indicated time points after oral glucose tolerance test (OGTT). G: Changes in blood glucose concentration at the indicated time points after IST. For the OGTT, mice were gavage fed with a 2 mg glucose/g body wt glucose load. For the IST, mice fasted for 6 h were intraperitoneally injected with 0.75 mU insulin/g body wt using an insulin syringe. OGTT and IST were performed on mice fasted for 6 h. Data are represented as mean ± SEM, n= 8/group. H: Immunoblot analysis of indicated proteins in WT and KO skeletal muscle and WAT. β-Tubulin was used as loading and transfer control. Representative blots are shown. I and J: Hyperinsulinemic-euglycemic clamp studies of Smad3-KO and WT mice. Glucose infusion rate required to maintained euglycemia in KO and WT mice (I). Blood glucose levels during the clamp study (J). K: Whole-body glucose uptake (peripheral insulin sensitivity). L: insulin-mediated suppression of hepatic glucose production rates (hepatic insulin sensitivity). Number represents percentage of suppression compared with basal hepatic glucose output. M: 2-DG uptake by soleus, gastrocnemius muscles, and WAT of KO and WT mice during clamp studies. Data are represented as mean ± SEM, n= 6/group. N and O: In vitro insulin-stimulated glucose uptake in skeletal muscles and WAT. 2-DG uptake into soleus muscle (N) and WAT (O) was measured for 30 min. The tissues from WT and KO were incubated in the absence [insulin(−)] or presence of insulin [insulin(+), 14 nmol/L], followed by measurement of 2-DG uptake. Net uptake of glucose was determined by subtracting the amount of [¹⁴C]mannitol from that of 2-DG. Data represent mean ± SEM, n= 10/group. *P < 0.05, **P < 0.01, ***P < 0.001 vs. WT controls.

Smad3-KO mice exhibit reduced physical activity. A–D: The mean total food (A), water (B), O₂ (C) consumptions, and CO₂ emission (D) during a 24-h light and dark cycle in WT and KO mice. Values in C and D were measured by indirect calorimetry and expressed as average Vo₂ and Vco₂ per kg body wt per h during a 24-h monitoring session of a light-dark cycle. E: Heat production of WT and KO mice was calculated from the O₂ production and the RER and expressed as average kcal/h/kg body wt. F: Fuel consumption was determined from the ratio of CO₂ emitted to the amount of O₂ consumed. Fuel type preference for carbohydrate has an RER of 1.0 versus fat of 0.7. G and H: Physical activity was assessed by the number of horizontal and vertical beam breaks, which represent the total horizontal (G) and rearing (H) movements during the 24-h monitoring period. I: Representative photograph showing the hind leg muscles of WT and KO mice. Small division on the ruler scale = 1 mm. J: Representative hematoxylin–eosin-stained histologic section of the WT and KO quadriceps muscles. Scale bars, 100 μm. K: Relative fold change in mRNA level of the indicated genes in WT and KO muscle was analyzed by real-time qPCR. Values were normalized to ribosomal protein L27. Results are represented as fold induction compared with WT. Primer sequences are summarized in Supplementary Table 1. Values represent mean ± SEM, n= 8/group. *P < 0.05, **P < 0.01, ***P < 0.001. (A high-quality color representation of this figure is available in the online issue.)

Smad3-KO adipose tissues have increased FA uptake and β-oxidation. A: Relative fold change in mRNA level of the indicated genes in WT and KO adipose tissue as determined by qPCR. Values are normalized to ribosomal protein L27. Normalized values from WT mice were arbitrarily assigned a value of 1. Only genes with twofold or greater change in expression are presented. Values represent mean ± SEM, n= 3/group. Primer sequences are summarized in Supplementary Table 1. B: Immunoblot analysis of indicated proteins in WT and KO adipose tissues. β-Tubulin was used as loading and transfer control. Representative blots from two mice for each genotype are shown. C–F: The rate of [¹⁴C]palmitate uptake (C), [¹⁴C]palmitate incorporation into triglycerides (D), lipolysis (E), and [¹⁴C]palmitate oxidation (F) at the indicated time points of adipocytes freshly isolated from the epididymal fat pads of WT and KO mice. Data are expressed as mean ± SEM, n= 3/group. *P < 0.05, **P < 0.01, ***P < 0.001 vs. WT controls.

Increased C/EBPβ-CHOP-10 interaction in Smad3-KO adipocytes. A: Schematic outline of procedure for in vivo coimmunoprecipitation, ChIP, and re-ChIP. B: DNA-protein complexes from WT or KO adipocytes were immunoprecipitated (ChIP) with anti-C/EBPβ or preimmune IgG. Enrichment of the DNA fragment containing the C/EBP binding site on the endogenous PPARβ/δ, PPARγ2 promoter, or a control sequence upstream was evaluated by PCR. A second immunoprecipitation step (re-ChIP) was also performed with an anti-HDAC1-specific antibody (H). I = input chromatin before immunoprecipitation, − = negative control (without anti-HDAC1 antibody), M = DNA marker, IB = immunoblot. C: Real-time qPCR was performed on immunoprecipitates of C/EBPβ antibodies and normalized to input. Data are expressed as mean ± SEM, n= 5/group. ***P < 0.001. D: Western blot analyses were also performed on proteins after immunoprecipitation with anti-C/EBPβ antibody. Proteins were separated by SDS-PAGE, blotted, and probed with anti-CHOP-10 primary antibody as indicated. Bands were detected by chemiluminescence. Western blot control with β-tubulin antibody was performed using cell lysate before immunoprecipitation. E: Schematic diagram showing the repression of PPARβ/δ by C/EBPβ is relieved on upregulation of CHOP-10 resulting from depletion of Smad3.

Smad3-KO mice are resistant to HFD-induced obesity and hepatic steatosis. A and B: Graph shows mean body weight of WT and KO mice on HFD or LFD over 18 weeks (A) and at the end of the 18-week diet regimen (B). C: Representative images of subcutaneous, epididymal, and visceral adipose tissues, and the liver of WT and KO mice from the two diet regimens. Small division on the ruler scale = 1 mm. D: Mean weights of the liver and the indicated adipose tissues of WT and KO mice after 18 weeks on HFD or LFD. Values are expressed as percentage of body weight. E: Representative hematoxylin–eosin-stained histologic section of the WT and KO EAT after 18 weeks on HFD or LFD treatment. Scale bars, 100 μm. F: Mean cross-sectional area of adipocytes (n= 400) from the WT and KO mice on the indicated diet regimen. G: Representative histologic section (oil red O and methylene blue staining) of the liver tissues. Scale bars, 100 μm. White arrows indicate oil droplets. H–J: Quantification of lipid accumulation in peripheral organs. Total triglyceride extracted from fresh-frozen liver (H), skeletal muscle (I), and pancreas (J) of WT and KO mice fed on HFD or LFD diet was expressed as amount in milimole per 1 g of protein. Data represent mean ± SEM, n = 10/group. ##P < 0.01 vs. WT LFD control; *P < 0.05, **P < 0.01, ***P < 0.001. EAT, epididymal adipose tissue; SAT, subcutaneous adipose tissue; VAT, visceral adipose tissue. (A high-quality digital representation of this figure is available in the online issue.)

Smad3 deficiency ameliorates HFD-induced insulin resistance and glucose intolerance. A–E: The mean plasma triglyceride (A), FFA (B), glycerol (C), blood glucose (D), and blood insulin (E) in WT and KO mice on HFD or LFD. Blood glucose and insulin levels were measured at 6-h fasted and fed states. F: Changes in blood glucose concentration at the indicated time points after OGTT. G: Changes in blood glucose concentration at the indicated time points after IST. OGTT and IST were performed on mice fasted for 6 h on an 18-week LFD or HFD regimen. Data represent mean ± SEM, n= 10/group. H–J: Hyperinsulinemic-euglycemic clamp studies of Smad3-KO and WT mice. Glucose infusion rate required to maintained euglycemia in KO and WT mice (H); whole-body glucose uptake (peripheral insulin sensitivity) (I); 2-DG uptake by soleus, gastrocnemius muscles, and WAT of HFD-fed KO and WT mice during clamp studies (J). Data are represented as mean ± SEM, n= 5/group. K–L: In vitro insulin-stimulated glucose uptake in skeletal muscles and WAT. 2-DG uptake into soleus muscle (K) and WAT (L) was measured for 30 min. The tissues from WT and KO were incubated in the absence [insulin(-)] or presence of insulin [insulin(+), 14 nmol/L], followed by measurement of 2-DG uptake. Net uptake of glucose was determined by subtracting the amount of [¹⁴C]mannitol from that of 2-DG. Data represent mean ± SEM, n = 6/group. *P < 0.05, **P < 0.01, ***P < 0.001 vs. WT controls. M: Immunoblot analysis of indicated proteins from skeletal muscle and WAT of HFD-fed WT and KO mice. β-Tubulin was used as loading and transfer control. Representative blots are shown.