GLP-1 (7-36 & 9-36)

ELISA ‘Total

Amide’

‘Active’

GLP-1 Physiology-

Publications with ‘Glucagon Like Peptide’ in Abstract- 2008 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.

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-

Food Intake

↑GLP-1

↑ Insulin & ↑ C-Peptide

↓ Food Intake

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-

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’ t1/2 ≈ 2min) → 9-36 and 9-37 (‘inactive’)

‘Active’

‘inactive’

DPP-4 mediated ‘inactivation’ of GLP-1.. From 7-36 and 7-37 → 9-36 and 9-37

The 7th & 8th Amino Acids (His & Ala) mitigate binding The major circulating biologically active form of GLP-1 is GLP-1 7-36 (~ ≥ 80%), with a lesser amounts of

the bioactive GLP-1 7-37 (~ < 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-1 9-36 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-

~80%

~20%GLP-1 (1-37)

GLP-1 (9-36amide )GLP-1 (7-36)

GLP-1 (7-37) GLP-1 (9-37amide )

→→

DPP-4

Active inactive

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly7 10 15 20 25 30 35

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-cellInsulin

+

↑GSIS- Insulin↓[Glucose]

α-cellGlucagon

o GLP-1 and the CNS & GI systems- ↓ [glucose] and ↓ food intakeo 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-

Publications with ‘Glucagon Like Peptide’ in Abstract- 2008 to October, 2009= 837 Why Measure= to measure DPP-4 activity or GLP-1 secretion.

GLP-1 Physiology-

GLP-1 Assay Devo. 2007-2008 Goal: Attain required specificity and improve sensitivity

Result: Sensitivity was reached, but interference was ubiquitous

GLP-1 Assay Devo. 2008-Fall 2009Goal 1: Keep specificity and the improved sensitivity

Goal 2: Fix linearity/InterferenceResult: Changed 1⁰ Ab (N-Term), reached goal