BioPath Online

Pathway Focus: Metabolic Disorders

Read Article 1
Interrogate the PPAR nuclear receptor family—LanthaScreen® TR-FRET PPAR Coactivator Assays | Read More
Read Article 2Measuring insulin and its receptors using ELISA—ELISA Kits for Metabolic Disorders | Read More
Read Article 3Study impacts on insulin resistance—Recombinant IGF-1 | Read More
Read Article 4Akt kinase signaling cascade in type 1 diabetes—Antibodies for Akt Signaling | Read More
New Antibodies

New Immunoassays

New Molecular Probes® Products

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).

Figure 1. Comparison of ligand binding affinities between PPAR isoforms. Serial dilutions of either the PPAR Γ agonist GW1929 or the PPAR α agonist GW7647 were assayed for their ability to competitively displace a fluorescent ligand (FluormoneTM Pan-PPAR green) from the GST-tagged LBD of PPAR α, PPAR δ, or PPAR Γ.


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.

Figure 3.  Analysis of ligand potencies using GeneBLAzer®PPAR Γ-UAS-bla HEK 293H cells. PPAR Γ cell-based assays were performed with serial dilutions of selected ligands. Cells were incubated with compounds for 20-24 hours before loading with the LiveBLAzerTM-FRET B/G substrate.




Product Cat. No.
 
PPAR αPV4892
PPAR δPV4893
PPAR Γ
PV4894
PPAR Γ cell-based assay
K1701
LiveBLAzer™-FRET B/G substrate
K1095

Measuring Insulin and its Receptors Using ELISA

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.


ProductSpeciesQty.
 Cat. No.
 
Insulin ELISA Kit
Hu
96 testsKAQ1251
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
Hu96 testsKHR9151
IR [pY972] ELISA  Kit
Hu96 testsKHR9141
IRS-1 total ELISA Kit
Hu, Ms, Rt
96 testsKHO0511
IRS-1 [PY612] ELISA Kit
Hu, Ms, Rt
96 testsKHO0931
IRS-1 [pS312] ELISA Kit
Hu, Ms, Rt
96 testsKHO0521
IGF-1R ELISA Kit
Hu, Ms, Rt
96 testsKHO0491
IGF-1R [pYpY1135/1136] ELISA Kit
Hu, Ms, Rt
96 testsKHO0501

Study IGF-1’s Impact On Insulin Resistance

Insulin-like growth factors (IGF) are structurally homologous to proinsulin. In addition to IGF-1’s role in development and cell growth, it is also a regulator of glucose metabolism. 

As its name suggests, IGF-1 (insulin-like growth factor-1) has similar properties to insulin.  IGF-1 binding to the extracellular domain of the insulin receptor (IR) and IGF-R leads to autophosphorylation of the receptor and activation of the intrinsic tyrosine kinase activity, which allows appropriate substrates to be phosphorylated.
IGF-1 shares 48% amino acid sequence identity with proinsulin and is capable of binding to the insulin receptor (IR), but with less affinity than insulin. 

IGF-1 levels play a key role in insulin resistance in Type II diabetes.  Recombinant IGF-1 can be used to study the impact of IGF-1 on insulin resistance.

ProductQty.
 Cat. No.
 
Recombinant Human IGF-1100 µg
PHG9071
Recombinant Human IGF-11 mg
PHG9073
Recombinant Human IGF-110 µg
PHG9074
Recombinant Human IGF-125 µg
PHG9075

Akt Kinase Signaling Cascade in Type 1 Diabetes

Akt/protein kinase B (Akt) and its signaling cascade are involved in number of different cellular functions, including glucose uptake, glycogen synthesis, cell growth, survival, apoptosis, protein synthesis, and endothelial nitric oxide production.  In Type 1 Diabetes, the kidney is exposed to fluctuating levels of blood glucose and exogenous insulin treatments. Both glucose and insulin can modulate Akt activity in various cell types and tissues, and thus can effect the signaling cascade and subsequent cellular processes.

Akt activation usually occurs via such  tyrosine kinase receptors as receptors for insulin and growth factors.  This activation initiates a cascade of events that help activate  mTOR involved in the regulation of protein synthesis.  mTOR facilitates protein translation through 4E-BP1, resulting in translation of specific mRNA subpopulations.
Enhanced protein synthesis may contribute to the development of renal hypertrophy via enhanced extracellular matrix accumulation, one of the key elements in diabetic nephropathy.

Invitrogen offers pan and phospho-site specific antibodies, which can check the activation status of AKT, mTOR, 4E-BP1, and other targets in their signaling cascade (Figures 1, 2). To make sure phospho-site–specific antibodies only detect activated antibodies, each lot of antibodies is tested with peptide blocking experiments, as well as stimulated versus unstimulated samples. With these tools, researchers can investigate the effect of glucose and exogenous insulin on cell functions and develop better treatments.

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.


TargetClonality, 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