Reversal of Obesity by Targeted Ablation of Adipose Tissue

Methods

We purchased C57BL/6 mice from Harlan Teklad, and Lep^ob/ob mice (stock 000632) from The Jackson Laboratory. All animal experiments involved standard procedures approved by The University of Texas M.D. Anderson Cancer Center and Baylor College of Medicine.

In vivo phage-display screening of a CX₇C library (C, cysteine; X, any amino acid residue)^[24,27] for fat-homing peptides was performed as described.^[24] In each biopanning round, an adult Lep^ob/ob female mouse was injected intravenously through the tail vein with 10¹⁰ transducing units (TU) of the library. Phage (~300 TU/g in round 1 increased to ~10⁴ TU/g in round 3) were recovered after 5 min of circulation from subcutaneous fat and bulk-amplified for each subsequent round. Phage amplified after the third round of panning were enriched for fat-specific binders by adapting an in vivo subtraction step:^[25] a lean, C57BL/6 female was injected intravenously with 10⁹ TU of phage selected in round 3. After 5 min, the unbound phage were recovered from circulation and amplified for the fourth and final round of biopanning in a Lep^ob/ob female mouse.

Staining of formalin-fixed, paraffin-embedded mouse tissue sections was performed as described.^[24] For phage-peptide immunolocalization, 10¹⁰ TU of CKGGRAKDC-phage or a control insertless phage was administered intravenously. Phage immunohistochemistry was carried out using a rabbit antibody to phage, B-7786 (Sigma), at 1:1000 dilution. For in vivo peptide homing validation, stock solutions of 5-carboxyfluorescein (FITC)-conjugated CKGGRAKDC and CARAC were chemically synthesized, cyclized and purified by high-performance liquid chromatography to 99% purity by AnaSpec. The solutions were prepared by dissolving the lyophilized peptides in DMSO to a concentration of 20 mM. Peptide-FITC solutions in phosphate buffer saline (PBS; 10 µl of 1 mM) were administered intravenously 5 min before tissue collection. For blood vessel localization, rhodamine-conjugated lectin-I (RL-1102, Vector Laboratories) was coadministered (10 µl of 2 mg/ml). Apoptosis was detected using standard TUNEL immunohistochemistry.^[41] Prohibitin immunolocalization was carried out with polyclonal rabbit antibody RDI-PROHIBITabr (Research Diagnostics) at 1:50 dilution. Immunohistochemistry was performed with the LSAB+ peroxidase kit (DAKO). Images were captured digitally with an Olympus IX70 microscope.

The high-calorie diet for obesity induction (TD97366: 25.4% fat, 21.79% protein, 38.41% carbohydrate) was purchased from Harlan Teklad. Mice were pre-fed with TD97366 before treatment to induce diet-related obesity until a weight greater than 45 g was acquired. Stocks of CKGGRAKDC-GG-_D(KLAKLAK)₂, CGDKAKGRC-GG-_D(KLAK-LAK)₂, _D(KLAKLAK)₂, CARAC-GG-_D(KLAKLAK)₂ and CKGGRAKDC were chemically synthesized, cyclized and purified by high-performance liquid chromatography to 99% purity (AnaSpec). They were prepared by dissolving lyophilized peptides in DMSO to a concentration of 65 mM. For each peptide, 100 nM of peptide stock dissolved in PBS was administered in the subcutaneous tissue of the back of C57BL/6 males daily for 4 weeks. Mouse weight, body temperature, and food and water consumption were monitored weekly. Tissue lipids were measured as described.^[42] Mice were fasted for 10 h for analyses of serum lipids and GTT (3 g glucose per kg body weight). The following kits were used: NEFA-C (WAKO Chemicals) for free fatty acids; GPO-TRINDER (Sigma) for glycerol and triacyl glyceride; rat insulin ELISA (Crystal Chemical) for insulin; TRINDER 100 (Sigma) for glucose; cholesterol E kit (WAKO chemicals) for cholesterol; and Quantikine M Immunoassay (R&D Systems) for leptin. Oxygen consumption and heat generation of fasting mice were measured for 24 h by indirect calorimetry with OXYMAX (Columbus Instruments). To quantify spontaneous activity, 8 mice (2 mice per cage) were placed in automated photocell activity cages (AccuScan Instruments). Mice were habituated to the activity cages before testing on the experimental day. Horizontal locomotor activity was computer-monitored overnight as the frequency of infrared beam breaks, which were recorded every hour for the duration of the test (14 h) using a Versamax Analyzer. Recordings were taken during the dark cycle lasting from 6 PM to 8 AM with water and food freely available.

In vitro biotinylation of the vasculature and extraction of membrane proteins was carried out as described.^[43] For expression and immobilization of GST fusion proteins on the column, we used the GST-Bind Kit (Novagen). Sepharose 4B (Amersham) was loaded with 100 µg of purified GST fusions desalted with Amicon filters (Millipore). Fat membrane proteins bound to GST fusion proteins were eluted with 1 mM FITC-peptides. For immunoblotting, polyclonal antibody to prohibitin, RDI-PROHIBITabr (RDI), diluted to 1:1000 was used. Sepharose 100EAH (Amersham) was loaded with 5 mg of CKGGRAKDC-GG-_D(KLAKLAK)₂ or CARAC-GG-D(KLAKLAK)₂. Chromatography was performed in an Econo Pump/Econo-Column Adaptor set (Biorad). Bound proteins were eluted by 100 mM glycine (pH 2.5). MALDI-TOF mass spectrometry was performed at a Core Facility of Baylor College of Medicine. To evaluate interaction of phage-CKGGRAKDC with prohibitin in vitro, binding of 10⁹ TU CKGGRAKDC-displaying or control (fd-Tet) phage to recombinant GST-fused prohibitin was performed as described.^[27] The polyclonal antibody to prohibitin, RDI-PROHIBITabr, diluted 1:10 was incubated with the immobilized protein for 1 h at room temperature before adding phage to test whether the binding would be blocked. Phage binding was assayed by infection of the host Escherichia coli and quantification of TU recovered from the wells.^[27]

We thank E. Koivunen, P. Dougherty, J. Cordella, S. Craig and C.D. Bucana for technical assistance; J. Folkman, H. Sage and J. Licinio for critical reading of the manuscript; and J. Trent, R. Benjamin, R.E. Pollock, J. Hannay, G. Fusaro and S. Chellappan for reagents.

Nat Med. 2004;10(6) © 2004 Nature Publishing Group

Cite this: Reversal of Obesity by Targeted Ablation of Adipose Tissue - Medscape - Jun 01, 2004.