Nutrient-dependent secretion of glucose-dependent insulinotropic polypeptide from primary murine K cells

Abstract

Aims/hypothesis: Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone with anti-apoptotic effects on the pancreatic beta cell. The aim of this study was to generate transgenic mice with fluorescently labelled GIP-secreting K cells and to use these to investigate pathways by which K cells detect nutrients.

Methods: Transgenic mice were generated in which the GIP promoter drives the expression of the yellow fluorescent protein Venus. Fluorescent cells were purified by flow cytometry and analysed by quantitative RT-PCR. GIP secretion was assayed in primary cultures of small intestine.

Results: Expression of Venus in transgenic mice was restricted to K cells, as assessed by immunofluorescence and measurements of the Gip mRNA and GIP protein contents of purified cells. K cells expressed high levels of mRNA for Kir6.2 (also known as Kcnj11), Sur1 (also known as Abcc8), Sglt1 (also known as Slc5a1), and of the G-protein-coupled lipid receptors Gpr40 (also known as Ffar1), Gpr119 and Gpr120. In primary cultures, GIP release was stimulated by glucose, glutamine and linoleic acid, and potentiated by forskolin plus 3-isobutyl-1-methylxanthine (IBMX), but was unaffected by the artificial sweetener sucralose. Secretion was half-maximal at 0.6 mmol/l glucose and partially mimicked by alpha-methylglucopyranoside, suggesting the involvement of SGLT1. Tolbutamide triggered secretion under basal conditions, whereas diazoxide suppressed responses in forskolin/IBMX.

Conclusions/interpretation: These transgenic mice and primary culture techniques provide novel opportunities to interrogate the mechanisms of GIP secretion. Glucose-triggered GIP secretion was SGLT1-dependent and modulated by K(ATP) channel activity but not determined by sweet taste receptors. Synergistic stimulation by elevated cAMP and glucose suggests that targeting appropriate G-protein-coupled receptors may provide opportunities to modulate GIP release in vivo.

Fig. 1

Generation of transgenic mice and confirmation of cell-specific Venus expression. a The BAC construct for making transgenic mice was made by cloning Venus into the coding region of Gip (for further details see ESM). b Colocalisation of direct Venus fluorescence (green) with GIP immunofluorescence (red) in the small intestine. Scale bar, 20 μm

Fig. 2

Collection and analysis of Venus-positive and control cells from the intestine. a, b Single-cell enzymatic digest of the upper small intestine from a GIP-Venus mouse, before (a) and after (b) FACS sorting. Differential interference contrast (DIC) images are shown on the left, with the corresponding fluorescence at 488 nm excitation on the right. After FACS sorting, >95% of the collected cells were Venus-positive. Scale bar, 30 μm. c K cells were collected from the upper small intestine by FACS sorting, using gates on pulse width and side and forward scatter to select single cells, and on yellow (580 nm) and green (530 nm) fluorescence to select either Venus-positive (R1) or Venus-negative cells. The excitation wavelength was 488 nm. d Histogram showing GIP protein content in unsorted, K_pos and L_pos cells from the upper small intestine. GIP content was analysed after cell extraction by ELISA. L_pos cells were FACS-sorted in a similar way to K_pos cells, using a transgenic mouse expressing Venus under the control of the proglucagon promoter [39]. Each bar represents three or four samples from different mice. **p<0.01, ***p<0.001 (Student’s t test) e, f, g Histograms showing relative gene expression of Gip, Gcg and Pyy in K_pos (black columns) and K_neg cells (white columns) from the upper small intestine. Expression was analysed by quantitative RT-PCR and compared with that of β-actin in the same sample. Three to six samples from separate mice were analysed for each bar. Data are presented as geometric mean, and the error bar was calculated from the log(base 2) data. Significance differences between K_pos and K_neg cells were found using Student’s t test; *p<0.05, ***p<0.001.

Fig. 3

Expression of candidate glucose-sensing machinery in K cells. a–f Histograms showing relative gene expression of K_ATP channel subunits (Kir6.2, Sur1), Gck, Sglt1, Glut2 and Glut5 in K_pos (black columns) and K_neg cells (white columns) from the upper small intestine. Expression was analysed by quantitative RT-PCR and compared with that of β-actin in the same sample. Each column represents three to six samples from separate mice. Data are presented as geometric mean, and the error bars were calculated from the log(base 2) data. Significance comparisons between K_pos and K_neg cells were calculated by Student’s t test; *p<0.05, **p<0.01, ***p<0.001. g Immunostaining for Venus (green) and SGLT1 (red) in the duodenum/jejunum, showing the apical localisation of SGLT1 on the villus. Because of the thickness of the optical slice (1.5 μm), only the apical tip of the K cell can be clearly seen. Scale bar, 20 μm

Fig. 4

Glucose-dependent GIP secretion from primary intestinal cultures. a Mixed intestinal cultures from the upper small intestine were incubated for 2–4 h in bath solution containing additions as indicated: control (Con), glucose (0.1, 1 or 10 mmol/l), glutamine (Gln, 10 mmol/l), fructose (Fruct, 10 mmol/l), tolbutamide (Tolb, 500 μmol/l) or αMG (10 mmol/l). Number of wells: glucose 0.1, 1, 10 mmol/l, 11, 13, 27, respectively; Gln, 12; Fruct, 9; Tolb, 19; αMG, 12. GIP was measured in the supernatant fraction and cell extracts, and is expressed relative to basal secretion measured in parallel on the same day (indicated by dashed line). Error bars represent 1 SE and significance is shown relative to baseline, analysed by single-factor t test; *p<0.05, **p<0.01. b Responsiveness of GIP secretion to a higher concentration of glucose (100 mmol/l) compared with sucralose (1 mmol/l), PMA (1 μmol/l) or fsk/IBMX (10 μmol/l of each). Number of wells: glucose, 13; sucralose, 5; PMA, 3; fsk/IBMX, 53. Experiments were carried out as in a. Error bars represent 1 SE, and significance is shown relative to baseline (shown by the dashed line) using a single-factor t test; **p<0.01, ***p<0.001

Fig. 5

Responses to lipids and expression of candidate G-protein coupled receptors in K cells. a GIP responses to fatty acids. Mixed intestinal cultures from the upper small intestine were incubated in bath solution containing additions as indicated: control (Con), oleoylethanolamide (OEA 10 μmol/l), linoleic acid (Linol, 100 μmol/l), fsk/IBMX (10 μmol/l of each). Number of wells: OEA, 9; Linol 11; fsk/IBMX, 9; fsk/IBMX+OEA, 6; fsk/IBMX+Linol, 9. GIP was measured in the supernatant fraction and cell extracts, and is expressed relative to basal secretion measured in parallel on the same day (indicated by dashed line). Error bars represent 1 SE, and significance is shown relative to baseline, analysed by single-factor t test. **p<0.01, ***p<0.001. Significance of differences between different combinations that included fsk/IBMX was tested by regression analysis. †††p<0.001. Gene expression of Gpr40 (b), Gpr119 (c) and Gpr120 (d) in K_pos (black columns) and K_neg cells (white columns) from the upper small intestine relative to that of β-actin as measured by quantitative RT-PCR. Each column represents three or four samples. Data are presented as geometric mean, and the error bar was calculated from the log(base 2) data. Significances of comparisons between K_pos and K_neg cells were calculated by Student’s t test performed on the log(base 2) data. **p<0.01, ***p<0.001

Fig. 6

GIP secretion in the presence of elevated cAMP. a Mixed intestinal cultures from the upper small intestine were incubated for 2-4 h in bath solution containing fsk/IBMX (10 μmol/l of each) plus different glucose concentrations, as indicated. GIP was measured in the supernatant fraction and cell extracts, and is expressed relative to the secretion in wells containing fsk/IBMX measured in parallel on the same day. The data were fitted with a logistic equation [y=A+(1−A)/(1+(x/ED₅₀)ⁿ)], with a maximal amplitude A=2.1, an ED₅₀=0.6 mmol/l and slope factor n of 1.3. Error bars represent 1 SE, and significance is shown relative to fsk+IBMX, analysed by single-factor t test; *p<0.05, **p<0.01. b GIP responses to agonists in the presence of fsk/IBMX (10 μmol/l of each). Experiments were carried out as in a, with additions as indicated: control (Con), glucose (Gluc, 10 mmol/l), glutamine (Gln, 10 mmol/l), tolbutamide (Tolb, 500 μmol/l), sucralose (Sucrl, 1 mmol/l), αMG (10 mmol/l), phloridzin (Phlorid, 5 μmol/l), diazoxide (Diazox, 340 μmol/l). Number of wells: Gluc, 17; Gln, 6; Tolb, 20; Sucrl, 3; αMG+Tolb, 13; αMG, 24; αMG+Phlorid, 6; aMG+Diazox, 6. Secretion was normalised to that measured in fsk/IBMX alone, measured in parallel on the same day (indicated by the dashed line). Error bars represent 1 SE, and significance is shown relative to fsk/IBMX, analysed by a single-factor t test; *p<0.05, **p<0.01, ***p<0.001. Significance of differences between different combinations that included αMG were tested by Student’s t test; ns non significant, †††p<0.001