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GLP-1 (7-36 & 9-36) ELISA ‘Total Amide’ ‘Active’.

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Presentation on theme: "GLP-1 (7-36 & 9-36) ELISA ‘Total Amide’ ‘Active’."— Presentation transcript:

1 GLP-1 (7-36 & 9-36) ELISA ‘Total Amide’ ‘Active’

2 GLP-1 Physiology-  Publications with ‘Glucagon Like Peptide’ in Abstract to October, 2009= 837  Why Measure= to measure DPP-4 activity or GLP-1 secretion.  What it is and What it does..  How and Where it acts..  When is it elevated and when decreased…  Who does and Why they would want to measure?  Relevant data.

3  Glucagon Like Peptide-1- (GLP-1) is mostly synthesized and secreted in response to nutrient ingestion (Carbohydrate, protein & lipids) from the intestinal L-Cells and plays multiple roles in metabolic homeostasis in response to food intake.  Levels of circulating [GLP-1] rise substantially on food intake fasting= ↓[GLP-1] fed state = ↑ [GLP-1] What it is- GLP-1 Physiology- What it is- GLP-1 Physiology- Food Intake ↑ GLP-1 ↑ Insulin & ↑ C-Peptide ↓ Food Intake

4  Only one GLP-1 Receptor (GLP1R) has been identified and is found  expressed in pancreatic islets (duct cells & β-cell ?α-cells), heart, kidney, CNS & the GI tract.  GLP-1 Stimulates Insulin Secretion, regulates food intake and regulates [Glucose] What it is- GLP-1 Physiology- What it is- GLP-1 Physiology-  GLP-1 is derived from prohormone convertase (PC1/3) cleavage of pro-glucagon to generate a 1-37 AA peptide.  Very little is known about the biological activity of GLP-1 (1-37).  1-37 is further process to form the secreted forms:  7-36 and 7-37 (‘active’ t 1/2 ≈ 2min) → 9-36 and 9-37 (‘inactive’) ‘Active’ ‘inactive’

5  DPP-4 mediated ‘inactivation’ of GLP-1.. From 7-36 and 7-37 → 9-36 and 9-37 The 7 th & 8 th Amino Acids (His & Ala) mitigate binding  The major circulating biologically active form of GLP-1 is GLP (~ ≥ 80%), with a lesser amounts of the bioactive GLP (~ < 20%).  Dipeptidyl Peptidase-4 (DPP-4, aka CD26) is a ubiquitous peptidase functions in two ways. The first is to bind adenosine deaminase and convey intracellular signals, largely in T-cells The second enzymatic function, when membrane bound and in circulation, DPP-4 has catalytic activity that mediates the cleavage of ‘active’ GIP, GLP-1 & GLP-2 to ‘inactive’ forms  Some studies have suggested that GLP may be an antagonist (block) of GLP-1 action, but other studies have failed to reproduce this data….  Other studies have identified alternative activities for 9-36/37  The rapid inactivation of these peptides by DPP-4-mediated cleavage poses important challenges for therapeutic efforts directed at enhancing GIP and GLP-1 activity in vivo. Multiple therapeutic drugs and targets revolving around either elevating GLP-1 secretion and/or inhibiting DPP-4 activity, with the goal of increasing the overall ‘active’ [GLP-1] What it is- GLP-1 Physiology- What it is- GLP-1 Physiology-

6  GLP-1 and islet-↓ [glucagon] and ↑ [insulin]  incretin response- GLP-1 is important in stimulating β-cell insulin secretion GLP-1 → ↑β-cell function ↑ β-cell glucose stimulated insulin secretion (GSIS) ↓ overall β-cell stress as well as ER stress = (↑β-cell function)  GLP-1 → ↑β-cell mass ↑ β-cell proliferation & differentiation ↓ apoptosis and/or β-cell death  GLP-1 → ↓ glucagon secretion from α-cells, which further lowers blood glucose = (↓ HGO)  preliminary evidence demonstrates that α-cells (Mix α/βcell ?) can be induced to secrete GLP-1 (through ↑ PC1/3 expression).  ─ ┤ Glucagon receptors (multiple therapeutics targets) and ↑ GLP (active) secretion What it does- GLP-1 Physiology- β- cell Insulin + ↑ GSIS- Insulin ↓ [Glucose] ­ α- cell Glucagon

7 o GLP-1 and the CNS & GI systems- ↓ [glucose] and ↓ food intake o GLP-1 is also synthesized in the brain stem and through axonal networks, delivered to the hypothalamus. Central and Peripheral GLP-1 acts in the CNS = (↑ satiety) o Numerous GLP-1 receptors are present in the CNS which can lead to ↓ in food intake (↑ satiety), and may control glucose flux in the liver and muscle as well as ↓ lipid storage in peripheral adipose tissue o ─ ┤ Gastric Emptying = (↑ satiety) o (?) Several studies have suggested that treatment with GLP-1 receptor agonists ( Incretin Mimetics ) &/or DPP4 Inhibitors may be associated with ↑ insulin sensitivity o (?, GLP-1 modulate insulin binding to insulin receptors) o GLP-1 action on CV System?? What it does- GLP-1 Physiology-

8  Publications with ‘Glucagon Like Peptide’ in Abstract to October, 2009= 837  Why Measure= to measure DPP-4 activity or GLP-1 secretion. GLP-1 Physiology-

9 GLP-1 Assay Devo Goal: Attain required specificity and improve sensitivity Result: Sensitivity was reached, but interference was ubiquitous

10 GLP-1 Assay Devo Fall 2009 Goal 1: Keep specificity and the improved sensitivity Goal 2: Fix linearity/Interference Goal 2: Fix linearity/Interference Result: Changed 1⁰ Ab (N-Term), reached goal


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