GIP (human)

Preparation Method

The cells were grown and transfected as described in GIP Receptor Signaling.125I labeled GIP was used as radioligand, and the competition binding analysis was performed on intact cells, seeded at a concentration of 50,000 cells/well, on the second day after transfection, the cells were incubated for 16 h at 4°C in 0.25 ml of buffer consisting of 25 mM Tris HCl, pH 7.4, and 5 mM MgCl2, using 15 pM 125I-GIP as radioligand. Increasing concentrations of unlabeled GIP, ranging from 10-11 to 10-6 M, were used as competitors. The competition binding was terminated by washing the cells once with 1 ml of binding buffer, and subsequently, the cells were lysed by the addition of 1 ml of lysis buffer (8 M carbamide, 3 M acetic acid, 2% Nonidet-P 40). The binding data were analyzed and IC50 values determined using nonlinear regression analysis.

Reaction Conditions

16 h at 4°C

Applications

For the homologous competition binding, IC50 value of GIP was observed to be 5.2 nM.

Cell lines

3T3-L1 cells

Preparation Method

Following incubation of 3T3-L1 cells, for the time periods indicated in figure legends, GIP in the incubation medium were precipitated with 1/10 vol. of 7% (wt/vol) ZnSO4, incubated on ice for 10 min, and 1/5 vol. 0.1 m NaOH added at room temp. The mixture was centrifuged and the supernatant assayed for glycerol with an enzymatic assay.

Reaction Conditions

10 min

Applications

GIP stimulated glycerol release with an EC50 of 3.28 +/- 0.63 Nm.

Animal models

Male Wistar rats (390–440 g, 12 wk old)

Preparation Method

GIP was dissolved in 0.9% NaCl containing 1% HSA and infused into the arterial line with the use of a syringe pump to give final perfusate concentrations of 1 nM GIP. After a basal period, peptides were infused alone for 10-min periods separated by 15-min rest periods, during which time endocrine secretion returned to basal levels.

Dosage form

1 nM GIP

Applications

Perfusion of the pancreas with 1 nM GIP increased insulin secretion significantly (P<0.001) relative to basal secretion during perfusion with 10 mM glucose alone.

References:

[1]. Deacon CF, Plamboeck A, Rosenkilde MM, de Heer J, Holst JJ. GIP-(3-42) does not antagonize insulinotropic effects of GIP at physiological concentrations. Am J Physiol Endocrinol Metab. 2006 Sep;291(3):E468-75.

[2]. McIntosh CH, Bremsak I, Lynn FC, Gill R, Hinke SA, Gelling R, Nian C, McKnight G, Jaspers S, Pederson RA. Glucose-dependent insulinotropic polypeptide stimulation of lipolysis in differentiated 3T3-L1 cells: wortmannin-sensitive inhibition by insulin. Endocrinology. 1999 Jan;140(1):398-404.