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To the editor:Binding between C-reactive protein (CRP) and human leptin in vitro, and inhibition by CRP of the effects of human leptin in mice in vivo, have recently been reported by Chen et al.1. In their in vitro studies the authors showed that after exposure of leptin-coupled agarose beads to serum and wash-ing, CRP was among the proteins eluted with acid. However, we have previously reported the specific calcium-dependent binding of CRP to agarose2,3. Therefore, we compared the binding of CRP from human serum to leptin-coupled agarose beads with that to identical beads that were blocked with Tris but had no coupled protein. We used the same coupling gel (Pierce AminoLink) as Chen et al.1 and tested both a normal (CRP 1 mg/liter) and acute-phase (CRP 69 mg/liter) human serum. We obtained identical trace quantities of CRP from the agarose beads regardless of the presence or absence of leptin. Furthermore, calcium chelation with EDTA eluted the same quantities of CRP from both sets of beads as did elution with acid, demonstrating that the CRP underwent its expected calcium-dependent binding to the agarose matrix2,3 independent of the presence of leptin. The nanogram quanti-ties of CRP that were eluted and detected by western blotting, as used by Chen et al.1, were consistent with the known binding of CRP to agarose and were directly related to the CRP concentration present in the serum. Chen et al.1 did not report the CRP concentration in the human serum they used1, but they also used rat serum, known to be rich in rat CRP (300 mg/liter or more), which binds more
avidly to agarose and is therefore recovered in higher yield4.
We then compared the binding of 125I-labeled leptin by CRP (ref. 2) to that by recombinant chimeric leptin recep-tor-Fc protein (R&D Systems), each cova-lently immobilized on CNBr-activated Sepharose (GE Healthcare). We used the same Sepharose matrix in two specific-ity controls: one bearing an equimolar amount of human serum amyloid P com-ponent (SAP)2, which is closely related to CRP, and one simply blocked with ethanol-amine alone. Identical, minimal traces of 125I-leptin remained associated with beads bearing CRP, SAP or no protein, in contrast to substantial specific binding by Sepharose beads coupled with leptin receptor (Table 1). In a separate experiment, we confirmed the functional integrity of the immobilized CRP and SAP, as evidenced by their capacity for specific calcium-dependent binding of, respectively, low-density lipoprotein5 and fibronectin6 from human serum.
We also investigated the effect of human CRP on the leptin-induced suppression of food intake and loss of body weight in age- and weight-matched groups of male wild-type C57BL/6 mice (Fig. 1). Twice-daily
intraperitoneal injections of recombinant human leptin (Calbiochem; 2.5 mg per kg body weight) produced the expected sup-pression of food intake and reduction in weight. Addition of six-fold molar excess of isolated pure human CRP (ref. 3; 70 mg/kg) to the same dose of leptin in each injection had no effect on the leptin-induced decrease in food intake or weight loss. Injection of CRP alone had no effect on food intake or body weight. At this dose of CRP, maximal and minimal circulating CRP concentra-tions were >500 mg/liter and ∼35 mg/liter, respectively, comparable (throughout the experiment) to major human acute-phase responses and much higher than both the concentration infused by Chen et al. and that in their human-CRP transgenic mice1. Although Chen et al.1 used ob/ob mice and infused leptin and CRP continuously rather than injecting them intermittently, our study in wild-type mice, using an effective dose of leptin and a substantial molar excess of human CRP (with plasma t½ = 3 4 h; ref. 7), did not show any inhibition of leptin func-tion by human CRP.
We conclude that the reported binding of CRP to human leptin in vitro1 was probably attributable to the known calcium-dependent
Table 1 Human leptin is not bound by human C-reactive protein
Protein immobilized on Sepharose Leptin bound per mol immobilized protein
Human C-reactive protein 0.0000448 mola
Human serum amyloid P component 0.0000405 mola
Leptin receptor-Fc chimera 0.383 molaNot different from binding to plain unsubstituted Sepharose. All binding was the same in the presence or absence of calcium or EDTA.
provide an important opportunity to test the relationships between leptin and CRP. We measured serum concentrations of CRP using a highly sensitive assay (Cardiophase hs-CRP, Dade Behring) in four patients with congenital leptin deficiency. Samples were taken when patients were clinically healthy and gave no history of recent exposure to infections or immunizations. Serum concen-trations of CRP were similar in congenitally leptin-deficient children and 20 age- and adiposity-matched obese children without leptin deficiency (means 4.2 (range 2.8–5.2) versus 3.9 (1.1–6.7) mg/liter, χ2 test for trend, P value nonsignificant: P > 0.05). We then examined the effects of subcutaneous recombinant human leptin on CRP concen-trations in children with congenital leptin deficiency. Mean concentrations of CRP were unchanged after 2 months (4.0 (1.4–6.5)
mg/liter) and 6 months (4.1 (0.4–9.2) mg/liter) of daily subcutaneous injections of recombinant human leptin. The bioactiv-ity of leptin in this experiment was evi-dent through its effect on ad libitum food intake at 2 months (mean reduction in food intake 64%, range 45–84%) and at 6 months (mean reduction in food intake 52%, range 40–65%)3,4. As very little weight was lost at the 2-month interval (mean 2.9 kg, range 1.5–5.2), it is unlikely that an independent effect of weight loss on CRP concentrations would explain these findings.
Our observations do not address the issue of whether the binding of leptin to CRP is a phy-siologically relevant event that might impair leptin action in some circumstances. Although we have no reason to question the observations of Chen et al. that leptin can increase CRP mRNA and protein levels in primary human
hepatocytes, our finding that leptin repletion in humans congenitally lacking leptin does not increase circulating CRP implies that such an effect is unlikely to be physiologically significant.
I Sadaf Farooqi & Stephen O’Rahilly
University Departments of Medicine and Clinical Biochemistry, Addenbrooke’s Hospital, Hills Road, Cambridge CB2 2QQ, UK.e-mail: [email protected]
COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.
1. Chen, K. et al. Nat. Med. 12, 425–432 (2006).2. Montague, C.T. et al. Nature 387, 903–908
(1997).3. Farooqi, I.S. et al. N. Engl. J. Med. 341, 879–888
(1999).4. Farooqi, I.S. et al. J. Clin. Invest. 110, 1093–1103
(2002).
NATURE MEDICINE VOLUME 13 | NUMBER 1 | JANUARY 2007 17
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CO R R E S P O N D E N C E
To the editor:Interaction of human leptin (hLEP) with human C-reactive protein (hCRP) was recently suggested as one of the major reasons for leptin resistance1. This notion is interesting because, if correct, preparation of leptin mutants that do not interact with CRP but retain their ability to interact and activate leptin recep-tors (LEPRs) would give us a potent biophar-maceutical reagent that could overcome such resistance. To validate this notion, we used sev-eral standard methods of determining protein:protein interactions.
In our first attempt to study the interac-tion between one recombinant and two native hCRPs (from three different sources) and hLEP (ref. 2), mixtures of the two proteins were prepared at various molar concentrations in 25 mM Tris-HCl; pH 8.0, or 10 mM HEPES
buffer (pH 7.4), containing 200 mM NaCl and 2 mM CaCl2, and resolved on a Superdex 75 column. There was a clear separation between CRP and hLEP (Supplementary Fig. 1 online), and there was no binding between the two pro-teins, as evidenced by the fact that there was no shift in the CRP peak (see Supplementary Fig. 1). Moreover, the size of the hLEP peak was not reduced relative to that obtained with the same concentration of hLEP injected alone. This experiment was repeated several times using all three CRPs and gave identical results. All CRPs appeared as a single peak that cor-responded to a molecular mass of ∼70 kDa and were eluted at a retention time of ∼13.8 min. As the anticipated molecular mass of the pentamer is 115 kDa, we checked whether this shift was owing to dissociation or retardation of the protein by the resin, the latter suggested
by the fact that the peak was skewed to the right (Supplementary Fig. 1). Accordingly, fractions corresponding to the CRP peak were collected and subjected to SDS-PAGE. To avoid the disassociation of CRP from pentamers into monomers, we dissolved the CRP in a sample buffer containing reducing agent but avoided boiling it (Supplementary Fig. 1). Using this procedure, we compared commercial CRP to that eluted from the Superdex column and found that the eluate consisted of only pen-tamers (Supplementary Fig. 1), whereas we identified traces of monomer in the com-mercial CRP (Supplementary Fig. 1). Because the premixing concentrations of CRP and leptin (625 µg/ml and 80 µg/ml, respectively) exceeded the physiological concentrations of CRP, we concluded that the existence of leptin:CRP complexes should be detectable under our
binding of CRP to the agarose matrix that was used. These results parallel the misidentifi-cation 30 years ago of SAP as ‘C1t’, a puta-tive fourth subcomponent of complement protein C1. SAP had been detected during affinity chromatography isolation of C1 from serum on immobilized IgG, but, in fact, it was present solely as a consequence of its avid calcium-dependent binding to the agarose matrix8 and had no interaction with C1 or any role in its function9. Inclusion of appropriate controls and awareness of the ligand-binding properties of the pentraxins are essential in such studies. We also demonstrate here that a substantial excess of CRP does not inhibit the effects of leptin on food intake and body weight in wild-type mice. Neither our obser-vations nor the 10,000-fold dynamic range of human plasma CRP (0.05–500 mg/liter)10
and its rapidity in the acute-phase response support the idea of an in vivo role for CRP in the modulation of leptin function.
Winston L Hutchinson1,3, Anthony P Coll2,3, J Ruth Gallimore1, Glenys A Tennent1 & Mark B Pepys1
1Centre for Amyloidosis and Acute Phase Proteins, Department of Medicine, University College London, Rowland Hill Street, London NW3 2PF, UK. 2Department of Clinical Biochemistry, University of Cambridge, Addenbrooke’s Hospital, Hills Road, Cambridge CB2 2QQ, UK. 3These authors contributed equally to this work.e-mail: [email protected]
ACKNOWLEDGMENTSSupported by grants to M.B.P. from the Medical Research Council (program grant G7900510) and the National Institutes of Health NHLBI Inflammation
and Thrombosis Program (project grant 1 R01 HL078578 01). A.P.C. is a Medical Research Council Clinician Scientist.
COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.
1. Chen, K. et al. Nat. Med. 12, 425–432 (2006).2. Pepys, M.B., Dash, A.C. & Ashley, J. Clin. Exp.
Immunol. 30, 32–37 (1977).3. de Beer, F.C. & Pepys, M.B. J. Immunol. Methods 50,
17–31 (1982).4. de Beer, F.C. et al. Immunology 45, 55–70 (1982).5. de Beer, F.C. et al. J. Exp. Med. 156, 230–242
(1982).6. de Beer, F.C., Baltz, M.L., Holford, S., Feinstein, A. &
Pepys, M.B. J. Exp. Med. 154, 1134–1149 (1981).7. Baltz, M.L., Rowe, I.F. & Pepys, M.B. Clin. Exp.
Immunol. 59, 243–250 (1985).8. Pepys, M.B. et al. Lancet 1, 1029–1031 (1977).9. Painter, R.H. J. Immunol. 119, 2203–2205 (1977).10. Pepys, M.B. & Hirschfield, G.M. J. Clin. Invest. 111,
1805–1812 (2003).
Figure 1 Effect of CRP on leptin action in mice. Each point represents mean ± s.d. (n = 6 mice per group). Open squares, vehicle (Tris/saline pH 8.0 containing 2 mM CaCl2); closed squares, leptin; open circles, CRP; closed circles, leptin and CRP. (a) Body weight. Significant differences on days 3–5 between effects of leptin versus vehicle, and between those of leptin plus CRP versus vehicle (P < 0.001 on days 3–5). (b) Daily food intake. Significant differences on days 3–5 between effects of leptin versus vehicle (P < 0.001 on day 3, P < 0.05 on day 4, P < 0.01 on day 5), and between effects of CRP plus leptin versus vehicle (P < 0.001 on day 3, P < 0.05 on days 4 and 5). (c) Cumulative food intake. Significant differences on days 4 and 5 between effects of leptin versus vehicle, and between effects of CRP plus leptin versus vehicle (P < 0.05 on day 4, P < 0.0001 on day 5). No significant differences between effects of vehicle versus CRP or between effects of leptin alone versus leptin plus CRP for any measurement on any day (P > 0.2). Significance was computed by a two-way analysis of variance (ANOVA).
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