Fig. 1. Antiinflammatory effects of CTRP-3 on the proinflammatory activation of adipocytes. Differentiated 3T3-L1 adipocytes (n = 9) were used for each of the stimulation experiments with LPS (20 ng/ml), lauric acid (100 μm), Pam3Cys (10 ng/ml), and poly(I:C) (10 ng/ml). Supernatant MCP-1 concentrations (picograms per milliliter) were measured by ELISA. Human recombinant CTRP-3 was added 30 min before simulation. Box plots indicate mean values ± sem. A, Effects of increasing doses of CTRP-3 (0.1, 1.0, and 10 μg/ml) on LPS-induced MCP-1 release. B, Effects of CTPR-3 on lauric acid-induced MCP-1 release. C, Effects of CTRP-3 on poly(I:C)-induced MCP-1 release. D, Expression of CTRP-3 was analyzed by Western blot analysis during differentiation of preadipocytes into mature adipocytes. The expression of β-actin was investigated as a housekeeping protein. From: C1q/TNF-Related Protein-3 Represents a Novel and Endogenous Lipopolysaccharide Antagonist of the Adipose Tissue Endocrinology. 2010;151(11):5267-5278. doi:10.1210/en.2010-0571 Endocrinology | Copyright © 2010 by The Endocrine Society

Fig. 2. Effects of siRNA-mediated knockdown of CTRP-3 on chemokine/adipokine release and cellular characteristics of adipocytes. A, Supernatant MCP-1 concentrations of 3T3-L1 adipocytes transfected with control siRNA or CTRP-3-specific siRNA, respectively. MCP-1 (pg/ml) was measured in supernatants of mature 3T3-L1 adipocytes by ELISA, and n = 9 wells were investigated per experimental group. Box plots indicate mean values ± sem. B, Supernatant adiponectin concentrations of 3T3-L1 adipocytes transfected with control siRNA or CTRP-3-specific siRNA, respectively. Adiponectin (nanograms per milliliter) was measured in supernatants of mature 3T3-L1 adipocytes by ELISA, and n = 16 wells were investigated per experimental group. Box plots indicate mean values ± sem. C, Demonstration of effective CTRP-3 knockdown by specific siRNA (Western blot). The specific knockdown of CTRP-3 in adipocytes was shown by Western blot analysis of CTRP-3 expression after transfection with control siRNA or CTRP-3-specific siRNA, respectively. The results of three independent experiments are depicted. β-Actin expression was investigated as a housekeeping protein. D, Morphological changes by Oil Red O staining of adipocytes transfected with control siRNA and CTRP-3 siRNA. Reduction of intracellular lipid content (red color) and lipid droplet size after siRNA-mediated knockdown of CTRP-3. E, Mean lipid droplet size and intracellular triglyceride concentration of adipocytes transfected with control siRNA and CTRP-3 siRNA. The mean lipid droplet diameter of n = 23 lipid droplets per experimental group is shown on the left. The ratio [arbitrary units (a.u.)] of mean intracellular triglyceride concentration/intracellular total protein concentration (n = 6 wells per experimental group) is shown on the right. From: C1q/TNF-Related Protein-3 Represents a Novel and Endogenous Lipopolysaccharide Antagonist of the Adipose Tissue Endocrinology. 2010;151(11):5267-5278. doi:10.1210/en.2010-0571 Endocrinology | Copyright © 2010 by The Endocrine Society

Fig. 3. Antiinflammatory effects of CTRP-3 in primary human monocytes isolated from healthy controls and patients with type 2 diabetes. Supernatant chemokine concentrations of human primary monocytes after stimulation with LPS (1 μg/ml) or LPS (1 μg/ml) plus CTRP-3 (1 μg/ml) were measured by ELISA and are depicted as means ± sem. Monocytes of 20 healthy controls (left) and 30 patients with type 2 diabetes mellitus (right) were investigated (for characteristics of the study population, please refer to Table 1). *, Comparison between stimulation groups (LPS vs. LPS + CTRP-3); **, comparison of LPS stimulation between healthy controls and diabetic patients; ***, comparison of LPS/CTRP-3 costimulation between healthy controls and diabetic patients. Mean values ± sem are shown. A, Supernatant concentrations of MIF. *, P = 0.003; ***, P < 0.001. B, Supernatant concentrations of MCP-1. *, P = 0.03 (left); *, P < 0.001 (right); **, P < 0.001; ***, P < 0.001. C, Supernatant concentration of CCL4. *, P < 0.001; ***, P < 0.001. D, Supernatant concentration of CCL3/MIP1α. ***, P < 0.001. From: C1q/TNF-Related Protein-3 Represents a Novel and Endogenous Lipopolysaccharide Antagonist of the Adipose Tissue Endocrinology. 2010;151(11):5267-5278. doi:10.1210/en.2010-0571 Endocrinology | Copyright © 2010 by The Endocrine Society

Fig. 4. CTRP-3 antagonizes lauric acid-induced proinflammatory activation of primary human monocytes. The effect of CTRP-3 (1 μg/ml) on lauric acid-induced (200 μm) release of IL-6 (picograms per milliliter) and TNF (picograms per milliliter) in primary human monocytes (n = 4 wells) isolated ex vivo was investigated by ELISA. A and B, The results of monocytes isolated from two (A and B) representative healthy and nondiabetic individuals are shown. Box plots indicate mean values ± sem. C, Expression of CTRP-3 in primary human adipocytes (positive control) and primary human monocytes and in the monocytic human cell line THP-1 by Western blot analysis. The expression of glyceraldehyde-3phosphate dehydrogenase (GAPDH) was investigated as a housekeeping protein. From: C1q/TNF-Related Protein-3 Represents a Novel and Endogenous Lipopolysaccharide Antagonist of the Adipose Tissue Endocrinology. 2010;151(11):5267-5278. doi:10.1210/en.2010-0571 Endocrinology | Copyright © 2010 by The Endocrine Society

Fig. 5. CTRP-3 specifically antagonizes the binding of bio-LPS to a designed Flag-tag TLR4/MD-2 fusion protein (LPS trap). A, ELISA-based system using a TLR4/MD-2 fusion protein. The binding of bio-LPS to the designed Flag-tag TLR4/MD-2 fusion protein (LPS trap) was investigated by an ELISA-based (biotin-streptavidin-peroxidase) TLR4/MD-2 assay. The LPS trap was coated to plastic wells via anti-Flag antibody (M2). Bio-LPS strongly bound to the TLR4/MD-2 fusion protein (lane 1). A 100-fold excess of non-bio-LPS was used to demonstrate specific and competitive inhibition of bio-LPS binding to the LPS trap (lane 2). Four doses of CTRP-3 (lanes 3–6) were tested for their ability to specifically inhibit the binding of bio-LPS to the LPS trap. CTRP-3 alone without bio-LPS was used as a negative control (lane 7). CTRP-3 at 1 and 10 μg/ml significantly reduced bio-LPS binding, indicating a specific inhibition of LPS binding to the TLR4/MD-2 fusion protein by CTRP-3. B, Immunoprecipitation. The LPS trap was incubated with bio-LPS, bio-LPS plus 100-fold excess of unlabeled LPS (specific and competitive inhibition), and CTRP-3 alone (negative control). Increasing doses of CTRP-3 were used to compete effectively with LPS. The LPS/LPS trap complex was pulled down with streptavidin-agarose and Western blotted, and the blot was probed with an anti-Flag antibody (M2). CTRP-3 at 10 μg/ml significantly reduced the binding of LPS to the TLR4/MD-2 fusion protein. Please note that the sensitivity for detection of specific CTRP-3 binding using immunoprecipitation is lower when compared with the ELISA. From: C1q/TNF-Related Protein-3 Represents a Novel and Endogenous Lipopolysaccharide Antagonist of the Adipose Tissue Endocrinology. 2010;151(11):5267-5278. doi:10.1210/en.2010-0571 Endocrinology | Copyright © 2010 by The Endocrine Society

Fig. 6. ELISA-based analysis of LPS binding to CTRP-3 Fig. 6. ELISA-based analysis of LPS binding to CTRP-3. A, To exclude that CTRP-3 effects on LPS binding to TLR4/MD-2 are caused by binding of CTRP-3 to LPS itself, an ELISA-based detection system for bio-LPS was developed using CTRP-3-coated plates. Plates were coated with 10 μg/ml CTRP-3. As a control, uncoated plates were used. After blocking with PBS/20% fetal calf serum, rising concentrations of bio-LPS were added for 2 h. After washing the plates, detection was performed by streptavidin-HRP and TMB (OD 450–630 nm). As shown by the identical curves, there was no specific binding of bio-LPS to CTRP-3. B, To demonstrate effective binding of CTRP-3 to the plastic wells as a prerequisite for the experimental procedure, the presence of CTRP-3 on the plastic wells was documented by an ELISA-based system using a primary anti-CTRP-3 antibody and a secondary antibody. Detection was performed by streptavidin-HRP and TMB (OD 450–630 nm). From: C1q/TNF-Related Protein-3 Represents a Novel and Endogenous Lipopolysaccharide Antagonist of the Adipose Tissue Endocrinology. 2010;151(11):5267-5278. doi:10.1210/en.2010-0571 Endocrinology | Copyright © 2010 by The Endocrine Society

Fig. 7. Western blot analysis of signal transduction protein expression in adipocytes after stimulation with CTRP-3. Mature 3T3-L1 adipocytes were stimulated with 1 μg/ml CTRP-3 for 5, 10, 30, 60, and 120 min. The expression of phosphorylated and unphosphorylated signal transduction proteins was investigated by Western blot analysis. AMPK, AMP-activated protein kinase. From: C1q/TNF-Related Protein-3 Represents a Novel and Endogenous Lipopolysaccharide Antagonist of the Adipose Tissue Endocrinology. 2010;151(11):5267-5278. doi:10.1210/en.2010-0571 Endocrinology | Copyright © 2010 by The Endocrine Society