RBP4 induces cytokines in mouse and human macrophages. (A and B) Holo-RBP4 induces TNF-α, IL-6, and MCP-1 secretion from cultured RAW264.7 macrophages (A) and primary mouse macrophages (B). (C) Mouse apo-RBP4 (Apo) has a greater effect than holo-RBP4 (Holo) on secretion of TNF-α, IL-6, and MCP-1 from RAW264.7 macrophages. Retinol was rebound to apo-RBP4 as an additional control (add-back). (D) Induction of cytokine secretion in primary human macrophages after stimulation with human holo- or apo-RBP4 or add-back RBP4 relative to vehicle control. (E) Induction of TNF-α, IL-6, MCP-1, IL-1β, and PPARγ gene expression in primary human macrophages after treatment with holo- or apo-RBP4 or add-back RBP4 relative to the vehicle control. All RBP4 incubations were with 50 μg/ml for 24 h. Either PBS or vehicle (dialysate) was used as a control. AU, arbitrary units. Data are expressed as means ± standard errors (SE). For panels A and B, n = 3 per group; *, P < 0.05 versus vehicle by t test. For panel C, n = 3 per group; *, P < 0.05 versus vehicle; #, P < 0.05 versus apo-RBP4 by ANOVA with post hoc analysis. For panels D and E, n = 7 per group. *, P < 0.05 versus vehicle by ANOVA with Wilcoxon analysis.

RBP4 indirectly inhibits insulin signaling in adipocytes by stimulating proinflammatory cytokine secretion from macrophages. (A) Representative Western blot of total Akt and phosphorylated Akt (S473 and T308) in 3T3L1 adipocytes after direct treatment with holo-RBP4 (50 μg/ml for 24 h) and insulin stimulation (100 nM for 10 min). (B) Quantification of experiments shown in panel A (n = 4). All conditions were normalized to cells treated with PBS and stimulated with insulin. (C) Representative Western blot of total Akt and phosphorylated Akt (S473 and T308) from contact coculture of 3T3L1 adipocytes and RAW264.7 macrophages after treatment with holo-RBP4 (50 μg/ml for 24 h) and insulin stimulation (10 nM or 100 nM for 10 min). (D) Quantification of experiments shown in panel C (n = 3). All conditions were normalized to cells treated with PBS and stimulated with 10 nM insulin. *, P < 0.05 versus vehicle under the same insulin conditions; #, P < 0.05 versus other insulin conditions for no RBP4 by two-way analysis of variance (ANOVA) with post hoc analysis. (E) Representative Western blot of total Akt and phosphorylated Akt (S473) from noncontact Transwell coculture of 3T3L1 adipocytes and RAW264.7 macrophages after treatment with holo-RBP4 (50 μg/ml for 24 h) and insulin (100 nM for 10 min). (F) Quantification of exper-iments shown in panel E (n = 4). All conditions were normalized to cells treated with PBS and then stimulated with insulin. Macs, macrophages. *, P < 0.05 versus no RBP4 in the absence or presence of macrophages; #, P < 0.05 versus absence of macrophages with same RBP4 treatment by two-way ANOVA with post hoc analysis. (G) Representative Western blot of total Akt and phosphorylated Akt (S473) from contact coculture of 3T3L1 adipocytes and RAW264.7 macrophages after treatment with neutralizing antibodies (abs) (1 or 5 μg/ml as indicated) for 30 min prior to holo-RBP4 (50 μg/ml for 24 h) treatment and then insulin (100 nM for 10 min). (H) Quantification of experiments shown in panel G (n = 2). All conditions were normalized to cells treated with PBS and then stimulated with insulin. *, P < 0.05 versus no RBP4 with control IgG; #, P < 0.05 versus RBP4 treatment with control IgG. Data are expressed as means ± SE except for panel H, in which n = 2.

RBP4 activates proinflammatory signaling pathways in macrophages. (A) Western blot of total and phosphorylated IKKα/IKKβ from RAW264.7 macrophages treated with holo-RBP4 (50 μg/ml) for the indicated times. (B) Quantification of NF-κB promoter activity in RAW264.7 macrophages transfected with pNFκB-Luc reporter plasmid and then treated with holo-RBP4 (50 μg/ml for 18 h). LPS (10 ng/ml) was used as a positive control. AU, arbitrary units. Data are expressed as the mean ± SE and normalized to β-galactosidase (β-Gal) expression. *, P < 0.05 versus PBS. (C) Western blot of total and phosphorylated Akt, ERK, p38, and JNK from RAW264.7 cells that were treated with either apo- or holo-RBP4 (50 μg/ml) for the indicated times.

JNK and TLR4 at least partially mediate the effects of RBP4 on insulin stimulation and cytokine production. (A) Western blot of total and phosphorylated Akt in 3T3L1 adipocytes from direct contact coculture with RAW264.7 macrophages. Cells were pretreated with JNK or IKK inhibitor (1 or 10 μM for 30 min), and then human holo-RBP4 was added (50 μg/ml for 24 h) prior to insulin (100 nM for 10 min). (B) Quantification of experiments shown in panel A. All conditions were normalized to cells treated with PBS and then stimulated with insulin. Data are means ± SE. *, P < 0.05 versus insulin-stimulated (+) with no RBP4. (C) mRNA expression of TNF-α, IL-6, and MCP-1 in RAW264.7 macrophages treated with JNK inhibitor (1 or 5 μM) and RBP4 as in panel A. The control for RBP4 was dialysate, and the control for JNK inhibitor was dimethyl sulfoxide (DMSO). AU, arbitrary units. Data are means ± SE and normalized to GAPDH expression (n = 3 to 4). *, P < 0.05 versus no RBP4; #, P < 0.05 versus no JNK inhibitor; +, P < 0.05 versus RBP4 plus 1 μM JNK inhibitor, one-way ANOVA. In, inhibitor. (D) TNF-α, IL-6, and MCP-1 secretion from primary mouse macrophages stimulated with holo- or apo-RBP4 or add-back RBP4 (50 μg/ml for 24 h). Macrophages were harvested from either control mice with functional JNK1 and JNK2 or mice with JNK1 and JNK2 deleted (JNK1/2 KO). Data are means ± SE (n = 4). *, P < 0.05 versus vehicle (dialysate) treatment within the same genotype; #, P < 0.05 for JNK1/2 KO versus control with same treatment; +, P < 0.05 versus holo-RBP4 and add-back RBP4 treatment within same genotype by two-way ANOVA with post hoc analysis. (E) TNF-α and IL-6 secretion from primary mouse macrophages stimulated with human holo- or apo-RBP4 or add-back RBP4 (50 μg/ml for 24 h). Macrophages were harvested and pooled from either two wild-type mice (open circles) or two mice with TLR4 deleted (TLR4 KO) (closed circles), and the experiments were done in duplicate. The bars show the mean with individual values indicated by circles (n = 2). *, P < 0.05 versus vehicle (dialysate) treatment within the same genotype; #, P < 0.05 for TLR4 KO versus control with same treatment; +, P < 0.05 versus holo- and add-back RBP4 treatment within same genotype by two-way ANOVA with post hoc analysis.

The ratio of apo-RBP4 to holo-RBP4 is elevated in human subjects with insulin resistance but not altered by insulin-glucose infusion during a euglycemic-hyperinsulinemic clamp. Values in serum from lean insulin-sensitive (I.S.), lean insulin-resistant (I.R.), and obese insulin-resistant nondiabetic subjects before (closed circles) and after (open circles) euglycemic-hyperinsulinemic clamp are shown. RBP4 levels (μg/ml) were measured by mass spectrometry immunoassay (A), and total serum retinol (μM) was measured by HPLC (B). (C) The molar concentration of serum RBP4 and retinol was used to calculate the molar ratio of RBP4 to retinol in serum for each subject. Data are means ± SE, with individual values shown (n = 8 per group). *, P < 0.05 versus lean insulin-sensitive subjects both before and after euglycemic-hyperinsulinemic clamp, as determined by two-way ANOVA with post hoc analysis. #, P < 0.05 for retinol values in obese subjects versus the two lean groups combined. The lean groups were combined because there was no difference in retinol values between the lean groups. The retinol values for each subject before and after clamp were averaged for comparison of obese versus lean serum retinol values by t test.