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Pathway Focus: Metabolic Disorders
Tools to Interrogate the PPAR Nuclear Receptor Family and Develop New Potential Treatments for Metabolic Syndrome
|Type 2 diabetes mellitus (T2DM), a global epidemic, is closely tied to metabolic syndrome and its related cardiovascular risk factors—abdominal obesity, dyslipidemia, hypertension, and hyperglycemia. |
Agonists of peroxisome proliferator-activated receptors (PPARs) are often used in combination with other drugs for the treatment of metabolic syndrome. However, many of these drugs, such as the thiazolidinediones (TZDs), possess deleterious side effects, including significant weight gain and peripheral edema; some have been associated with increased cardiovascular risk. New efforts to develop safer and more selective treatments of T2DM include the search for selective modulators that bind distinctly to the ligand-binding pocket of PPARs, leading to alternative receptor conformations, differential cofactor recruitment/displacement, differential gene expression, and ultimately differential biological responses.
Biochemical tools to evaluate compound binding and coactivator recruitment to each isoform, along with cellular reporter assays, are discussed below.
LanthaScreen® TR-FRET PPAR Competitive Binding Assays
PPAR binding assays can screen compound libraries to identify tight binders and determine isoform selectivity. The general approach includes a terbium-labeled anti-GST antibody that indirectly labels a nuclear receptor (NR) by binding to its GST tag. Competitive binding to the NR is detected by a test compound’s ability to displace a fluorescent ligand (tracer) from the NR, which results in a loss of FRET signal between the Tb-anti-GST antibody and the tracer (Figure 1).
|LanthaScreen® TR-FRET PPAR Coactivator Assays|
PPAR coactivator assays can screen for compounds that recruit a particular coactivator peptide or determine the recruitment pattern of a set of coregulator peptides to a particular PPAR isoform upon binding to the compound. The assay uses a terbium-labeled anti-GST antibody to indirectly label a PPAR-LBD by binding to its GST tag. Recruitment of fluorescein-labeled coactivator peptides is detected by an increase in TR-FRET signaling between the Tb-anti-GST antibody and the fluorescein of the peptide, while displacement of corepressor peptides can be observed by a decrease in the TR-FRET signal (Figure 2).
Figure 2. Coregulator Peptide Profile for PPAR Γ. A panel of fluorescein-labeled coregulator peptides was screened against PPAR gamma in the presence and absence of a variety of ligands. Data for ligands is reported as the fold change of the TR-FRET signal of the receptor with ligand divided by receptor without ligand. Ligand independent recruitment is indicated in the “no ligand” data set where the TR-FRET signal of the receptor without ligand is divided by the no receptor control. Values greater than one indicate peptide recruitment while values less than one indicate displacement.
|GeneBLAzer® PPAR-UAS-bla HEK 293 cell lines|
PPAR cell-based assays screen libraries of compounds for agonists or antagonists, and also help determine if compounds are partial agonists or mixed agonists/antagonists. These cells contain a GAL4-DNA binding domain/PPAR ligand binding domain fusion transiently transduced via baculovirus (for PPAR alpha) or stably integrated (for PPAR gamma and delta) into CellSensor® UAS-bla HEK293 cells.
CellSensor® UAS-bla HEK293 cells contain a β-lactamase (bla) reporter gene under transcriptional control of an Upstream Activator Sequence (UAS). β-lactamase (BLA) expression is detected using a membrane-permeable FRET-based substrate, which allows measurement of activity in living cells; the dual emission wavelength read-out significantly reduces experimental variables (Figure 3). For more information on our PPAR alpha cell-based assay.
SelectScreen® Cell-based Nuclear Receptor Profiling Services
The SelectScreen® Cell-based Nuclear Receptor Profiling Service utilizes our comprehensive library of GeneBLAzer® target-specific nuclear receptor cell lines and our robust GeneBLAzer® beta-lactamase (bla) reporter technology. The service’s flexible approach to screening enables rapid profiling against a panel of nuclear receptor cell lines by EC50/IC50 determinations in both agonist (% activation) and antagonist (% inhibition) mode, using 10-point dose response curves. The service provides customers with a wide range of combinations for screening, from a small subset of compounds against multiple cell lines to many compounds on one cell line, such as a library screen.
|Product|| Cat. No.|
|PPAR Γ cell-based assay||K1701|
|LiveBLAzer™-FRET B/G substrate||K1095|
|Diabetes mellitus leads to high blood glucose levels due to defects in either insulin secretion or insulin action in the body. Insulin stimulates receptors including insulin receptor (IR), insulin receptor substrate-1 (IRS-1), insulin-like growth factor-1 receptor (IGF-1R), which results in a cascade of signaling events. Functionally, IR is thought to regulate metabolism, while IGF-1R mediates growth and differentiation. ||Invitrogen sandwich ELISA kits quickly detect and quantify specific proteins in normal and diseased models (Figures 1, 2). The ELISA kits allow results to be collected in an easy and reproducible fashion. Calibrated standard curves accurately quantify the level of protein in each experimental run. The ELISA technology allows a more detailed understanding of protein levels in metabolic disorders to ultimately develop therapies.|
|Figure 1. Detection of upregulated phosphorylation of IGF0-1R in treated cells. MCF 7 cells were pretreated with 1 mM sodium orthovanadate for 16 hours, then treated with IGF 1. Untreated MCF 7 cells were used as controls. Cell extracts were prepared and cell lysates were analyzed with IGF 1R [pYpY1135/1136] ELISA and Invitrogen™ IGF 1R ELISA. The results show that the phosphorylation of IGF 1R is upregulated in IGF 1 treated MCF 7 cells, whereas the level of IGF 1R remains consistant in IGF 1 treated and untreated controls.|
Figure 2. ELISA kits detect IR phosphorylation. CHO‑T cells were stimulated using 100 nM insulin for 10 minutes. Unstimulated cells were used as control. Cell lysates from the cells were measured for the levels of IR and phosphorylated IR. The results show that IR (b‑subunit) ELISA kit detects phosphorylated IR in insulin‑stimulated CHO‑T and non‑phosphorylated IR in unstimulated control cells.
|Product||Species||Qty.|| Cat. No. |
|Insulin ELISA Kit ||Hu||96 tests||KAQ1251|
|IR Total ELISA Kit ||Hu, Ms, Rt||96 tests||KHO1091|
|IR [pYpY1162/1163] ELISA Kit ||Hu||96 tests||KHO0741|
|IR [pY1158] ELISA Kit||Hu, Ms, Rt||96 tests||KHR9121|
|IR [pY1328] ELISA Kit||Hu||96 tests||KHR9151|
|IR [pY972] ELISA Kit||Hu||96 tests||KHR9141|
|IRS-1 total ELISA Kit||Hu, Ms, Rt||96 tests||KHO0511|
|IRS-1 [PY612] ELISA Kit||Hu, Ms, Rt||96 tests||KHO0931|
|IRS-1 [pS312] ELISA Kit||Hu, Ms, Rt||96 tests||KHO0521|
|IGF-1R ELISA Kit||Hu, Ms, Rt||96 tests||KHO0491|
|IGF-1R [pYpY1135/1136] ELISA Kit||Hu, Ms, Rt||96 tests||KHO0501|
|Product||Qty.|| Cat. No. |
|Recombinant Human IGF-1||100 µg||PHG9071|
|Recombinant Human IGF-1||1 mg||PHG9073|
|Recombinant Human IGF-1||10 µg||PHG9074|
|Recombinant Human IGF-1||25 µg||PHG9075|
|Figure 1. AKT/PKB [pS473] Rabbit Monoclonal Antibody western blot and peptide competition. Extracts of NIH3T3 cells unstimulated (lane 1) or stimulated with 50 ng/mL PDGF for 5 minutes (lanes 2–5) were resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF. The membrane was incubated with the Akt/PKB [pS473] monoclonal antibody for two hours following prior incubation with: no peptide (1, 2), the non-phosphorylated peptide corresponding to the phosphopeptide immunogen (3), a generic phosphoserine-containing peptide (4), or the phosphopeptide immunogen (5). The membrane was incubated with goat F(ab’)2 anti-rabbit IgG HRP-conjugate and signals were detected using the Pierce SuperSignal™ method. Only the phosphopeptide corresponding to Akt/PKB [pS473] completely blocks the antibody signal, demonstrating the specificity of the antibody. The data also show the induction of Akt/PKB [pS473] phosphorylation by the addition of PDGF in this cell system.|
|Figure 2. AKT/PKB [pS473], Rabbit Monoclonal Antibody immunofluorescence staining. Serum-starved NIH3T3 cells left untreated (left) or treated with PDGF (right) were fixed prior to immunostaining with the Akt [pS473] rabbit monoclonal antibody. The signal was detected with an anti-rabbit FITC conjugated secondary antibody. The antibody detected phosphorylated Akt in PDGF-treated NIH3T3.|
|Target||Clonality, Clone, (Isotype)||Reactive Species||Applications||Qty.|| Cat. No. |
|Akt ||pAb (Rb IgG)||Hu, Ms (Rt)||WB||200 μl||44609G|
|Akt ||mAB, 9Q7 (Ms IgG3)||Hu||WB||100 μg||AHO1112|
|Akt [pS473] ||mAb, (Rb IgG)||Hu, Ms (Rt)||WB, ICC||10 blot||44621G|
|Akt [pT308] ||pAb (Rb IgG)||Hu, Ms (Rt)||WB||10 blot||44602G|
|AKT/PKB1 [PS473] ||pAb (Rb IgG)||Hu, Ms||WB||100 µl ||44623G|
|mTOR||mAB 215Q18||Hu, Ms, Rt||WB||100 μg||AHO1232|
|mTOR [pS2448]||pAb (Rb IgG) ||Hu, Ms, H, (Rt)||WB, IF||10 blot||441125G|
|4E BP1||pAb, ARO-17 (Rb IgG)||Ms, Rt, (Hu)||WB, E||100 μg||512900|
|4EBP1||mAB, 554R16 (IgG1)||Hu, Ms, Rt||WB||100 μg||AHO1382|
|4E-BP1 [pT46]||pAb (Rb IgG)||Hu, Ms||WB||10 blot||441170G|