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. 2012 Oct 1;303(7):E899-907.
doi: 10.1152/ajpendo.00116.2012. Epub 2012 Aug 7.

Role of Fatty Acid Transport Protein 4 in Oleic Acid-Induced Glucagon-Like peptide-1 Secretion From Murine Intestinal L Cells

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Free PMC article

Role of Fatty Acid Transport Protein 4 in Oleic Acid-Induced Glucagon-Like peptide-1 Secretion From Murine Intestinal L Cells

M A Poreba et al. Am J Physiol Endocrinol Metab. .
Free PMC article

Abstract

The antidiabetic intestinal L cell hormone glucagon-like peptide-1 (GLP-1) enhances glucose-dependent insulin secretion and inhibits gastric emptying. GLP-1 secretion is stimulated by luminal oleic acid (OA), which crosses the cell membrane by an unknown mechanism. We hypothesized that L cell fatty acid transport proteins (FATPs) are essential for OA-induced GLP-1 release. Therefore, the murine GLUTag L cell model was used for immunoblotting, [(3)H]OA uptake assay, and GLP-1 secretion assay as determined by radioimmunoassay following treatment with OA ± phloretin, sulfo-N-succinimidyl oleate, or siRNA against FATP4. FATP4(-/-) and cluster-of-differentiation 36 (CD36)(-/-) mice received intraileal OA, and plasma GLP-1 was measured by sandwich immunoassay. GLUTag cells were found to express CD36, FATP1, FATP3, and FATP4. The cells demonstrated specific (3)H[OA] uptake that was dose-dependently inhibited by 500 and 1,000 μM unlabeled OA (P < 0.001). Cell viability was not altered by treatment with OA. Phloretin and sulfo-N-succinimidyl oleate, inhibitors of protein-mediated transport and CD36, respectively, also decreased [(3)H]OA uptake, as did knockdown of FATP4 by siRNA transfection (P < 0.05-0.001). OA dose-dependently increased GLP-1 secretion at 500 and 1,000 μM (P < 0.001), whereas phloretin, sulfo-N-succinimidyl oleate, and FATP4 knockdown decreased this response (P < 0.05-0.01). FATP4(-/-) mice displayed lower plasma GLP-1 at 60 min in response to intraileal OA (P < 0.05), whereas, unexpectedly, CD36(-/-) mice displayed higher basal GLP-1 levels (P < 0.01) but a normal response to intraileal OA. Together, these findings demonstrate a key role for FATP4 in OA-induced GLP-1 secretion from the murine L cell in vitro and in vivo, whereas the precise role of CD36 remains unclear.

Figures

Fig. 1.
Fig. 1.
Expression of fatty acid transport proteins in the L cell. Immunoblot for cluster of differentiation 36 (CD36) (55 kDa: nonglycosylated intracellular form; 88 kDa: glycosylated membrane form; A), fatty acid transport protein (FATP)1 (63 kDa; B), FATP3 (72 kDa; C), and FATP4 (72 kDa; D) in murine GLUTag L cells (n = 3). Actin (42 kDa) was used as the loading control and murine duodenum as a positive (+ve) control.
Fig. 2.
Fig. 2.
Oleic acid (OA) uptake in the L cell and the effect of OA on glucagon-like peptide-1 (GLP-1) secretion. A: GLUTag cells were incubated with [3H]OA and treated with vehicle control (solid line) or 500 (dashed line) or 1,000 μM (dotted line) unlabeled OA, followed by determination of [3H]OA uptake (●). [14C]mannitol was used as a cell integrity control in each treatment group (▲). Counts per minute (cpm) were normalized to total protein (inset: expanded scale) (n = 6). B: GLUTag cells were treated with vehicle (control) or increasing concentrations of OA for 2 h, and secretion of GLP-1 was determined by radioimmunoassay (n = 6–11). Basal secretion was 8.6 ± 0.8% of total cell content. C: GLUTag cells were treated for 2 h with vehicle alone (control), 5 mM H2O2, or 1,000 μM OA for 2 h, followed by determination of viability using neutral red uptake, as assessed by optical density (OD) at 540 nm (n = 8). *P < 0.05, **P < 0.01, and ***P < 0.001 vs. control; ##P < 0.01 and ###P < 0.001 for 500 vs. 1,000 μM unlabeled OA or as indicated.
Fig. 3.
Fig. 3.
Effect of phloretin on L cell OA uptake and GLP-1 secretion. A: GLUTag cells were incubated with [3H]OA and treated with vehicle control (solid line), 1,000 μM unlabeled OA (dotted line), or 200 μM phloretin (dashed line), followed by determination of [3H]OA uptake (●). [14C]mannitol was used as a cell integrity control in each treatment group (▲). CPM were normalized to total protein (inset: expanded scale) (n = 6). B: GLUTag cells were treated with either vehicle control or 200 μM phloretin and incubated further with or without 1,000 μM OA. GLP-1 secretion was determined by radioimmunoassay (n = 11–12). Basal secretion was 8.5 ± 1.4% of total cell content. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. respective control; ##P < 0.01 as indicated.
Fig. 4.
Fig. 4.
Role of CD36 in L cell OA uptake and GLP-1 secretion. A: GLUTag cells were incubated with [3H]OA and treated with vehicle control (solid line), 1,000 μM unlabeled OA (dotted line), or 400 μM SSO (dashed line), followed by determination of [3H]OA uptake (●). [14C]mannitol was used as a cell integrity control in each treatment group (▲). CPM were normalized to total protein (n = 6). B: GLUTag cells were treated with either vehicle control or 400 μM SSO and were incubated further with or without 1,000 μM OA. GLP-1 secretion was determined by radioimmunoassay (n = 11–12). Basal secretion was 8.1 ± 0.8% of total cell content. C: immunoblot for CD36 (55 kDa) and actin (42 kDa; loading control) in the ileum of control (CO) and CD36-null (KO) mice (representative of n = 5–6). D: OA (125 mM in 125 mM Tween-80) was injected directly into the ileum of control (solid line) and CD36-null (dashed line) mice, and blood samples were collected at t = 0 and 15 min or at t = 0 and 60 min. Total GLP-1 levels were determined in the collected plasma using a sandwich immunoassay (n = 9–19). *P < 0.05, **P < 0.01, and ***P < 0.001 vs. respective control; #P < 0.05 as indicated.
Fig. 5.
Fig. 5.
Role of FATP4 in L cell OA uptake and GLP-1 secretion. A: GLUTag cells were treated with scrambled or FATP4 small interfering RNA (siRNA), and FATP4 and actin (loading control) levels were detected by immunoblot (representative of n = 3). B: [3H]OA uptake was determined in GLUTag cells treated with scrambled (solid line) or FATP4 siRNA (dashed line) (●). [14C]mannitol was used as a cell integrity control in each treatment group (▲). CPM were normalized to total protein (n = 4–5). C: GLUTag cells were treated with scrambled or FATP4 siRNA and were incubated further with or without 1,000 μM OA. GLP-1 secretion was determined by radioimmunoassay (n = 7–8). Basal secretion was 6.7 ± 0.5% of total cell content. D: immunocytochemistry for FATP4 (red) in vehicle- (images a and c) and OA-treated GLUTag cells (images b and d); Blue represents nuclei (images a and b). Images a and b represent whole cell images, whereas the sections shown in images c and d were taken from 3-dimensional “z-stack” analyses at approximately the mid-cell level. E: immunoblot for FATP4 (72 kDa) and actin (42 kDa; loading control) in the ileum of wild-type (WT) and FATP4-null (KO) mice (representative of n = 5). F: OA (125 mM in 125 mM Tween-80) was injected directly into the ileum of WT (solid line) and KO (dashed line) mice, and blood samples were collected at t = 0 and 15 min or at t = 0 and 60 min. Total GLP-1 levels were determined in the collected plasma using an immunoassay system (n = 7–22). *P < 0.05 and **P < 0.01 vs. respective control; #P < 0.05 as indicated.

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