Long chain saturated and unsaturated fatty acids exert opposing effects on viability and function of GLP-1-producing cells: Mechanisms of lipotoxicity

Abstract

Background and aim: Fatty acids acutely stimulate GLP-1 secretion from L-cells in vivo. However, a high fat diet has been shown to reduce the density of L-cells in the mouse intestine and a positive correlation has been indicated between L-cell number and GLP-1 secretion. Thus, the mechanism of fatty acid-stimulated GLP-1 secretion, potential effects of long-term exposure to elevated levels of different fatty acid species, and underlying mechanisms are not fully understood. In the present study, we sought to determine how long-term exposure to saturated (16:0) and unsaturated (18:1) fatty acids, by direct effects on GLP-1-producing cells, alter function and viability, and the underlying mechanisms.

Methods: GLP-1-secreting GLUTag cells were cultured in the presence/absence of saturated (16:0) and unsaturated (18:1) fatty acids (0.125 mM for 48 h, followed by analyses of viability and apoptosis, as well as involvement of fatty acid oxidation, free fatty acid receptors (FFAR1) and ceramide synthesis. In addition, effects on the expression of proglucagon, prohormone convertase 1/3 (PC1/3), free fatty acid receptors (FFAR1, FFAR3), sodium glucose co-transporter (SGLT) and subsequent secretory response were determined.

Results: Saturated (16:0) and unsaturated (18:1) fatty acids exerted opposing effects on the induction of apoptosis (1.4-fold increase in DNA fragmentation by palmitate and a 0.5-fold reduction by oleate; p<0.01). Palmitate-induced apoptosis was associated with increased ceramide content and co-incubation with Fumonisin B1 abolished this lipo apoptosis. Oleate, on the other hand, reduced ceramide content, and-unlike palmitate-upregulated FFAR1 and FFAR3, evoking a 2-fold increase in FFAR1-mediated GLP-1 secretion following acute exposure to 0.125 mmol/L palmitate; (p<0.05).

Conclusion/interpretation: Saturated (16:0), but not unsaturated (18:1), fatty acids induce ceramide-mediated apoptosis of GLP-1-producing cells. Further, unsaturated fatty acids confer lipoprotection, enhancing viability and function of GLP-1-secreting cells. These data provide potential mechanistic insight contributing to reduced L-cell mass following a high fat diet and differential effects of saturated and unsaturated fatty acids on GLP-1 secretion in vivo.

Fig 1. Palmitate and oleate exert opposing effects on the formation of reactive oxygen species, activation of the mitogen-activated protein kinase p38 and viability of GLP-1-producing cells.

0.125 mM oleate significantly decreases—while 0.125 mM palmitate significantly increase—caspase-3 activation (A) and DNA fragmentation (B) in GLUTag cells following a 48h incubation. Co-incubation of 0.125mM palmitate with 0.125mM Oleate abolishes palmitate induced caspase-3 activity (C) and DNA fragmentation (D). ROS production in GLP-1-secreting cells after 48 h (E) and phosphorylation of the ROS-sensitive kinase p-38 following 8h (F) is increased in response to 0.125mM palmitate, but not in response to 0.125mM Oleate. Bars represent mean ± SEM for n = 3–6 independent experiments analyzed in duplicates. Comparisons between groups were made by a one-way ANOVA, and Student-Newman-Keul’s post hoc test. *, p<0.05; ***, p<0.001 compared with control cells. #, p<0.05 compared with palmitate-treated cells.

Fig 2. Palmitate-induced lipotoxicity in GLP-1-producing cells is dependent on the formation of ceramide.

Co-incubation of 0.125mM palmitate with etomoxir did not significantly alter caspase-3 activation (A) or viability (B) after 48 h. Similarly, co-incubations of 0.125 mM palmitate and FFAR1 antagonist GW1100 did not alter palmitate induced caspase-3 activity (C) or viability (D). GLUTag cells were stained using Hoechst (blue) and a ceramide monoclonal antibody (green) following exposure to 0.125mM palmitate and 0.125mM oleate for 6h (E), where 0.125mM palmitate—but not 0.125mM oleate—increasedthe number of ceramide positive GLUTag cells as assessed by detection of fluorescence proportional to the number of positive cells (F). Co-incubation of 0.125mM palmitate with Fumonisin B1 significantly attenuate palmitate induced caspase-3 activity (G) and reduced viability (H) following 48 h. Bars represent mean ± SEM for n = 3 independent experiment analyzed in duplicates. Comparisons between groups were made by a one-way ANOVA, and Student-Newman-Keul’s post hoc test. *, p<0.05 compared with control cells. #, p<0.05 compared with palmitate-treated cells.

Fig 3. Oleate, but not palmitate, increases the expression of G protein-coupled receptor FFAR1 mRNA and amplifies the acute secretory response of GLP-1 producing cells to fatty acids.

Whereas 0.125 mM palmitate significantly reduced GLUTag proglucagon and FFAR1 (GPR40) / FFAR3 (GPR43) mRNA expression after 48h (A), oleate exposure significantly increased the expression of proglucagon / FFAR 1 (GPR40) and FFAR3 (GPR43) mRNA after 24h / 48h respectively (B). GLP-1 secretion in response to 0.5 mM palmitate was increased 2-fold following a 48h exposure to 0.125mM oleate (C). Comparisons between groups were made by a one-way ANOVA, and Student-Newman-Keul’s post hoc test. Bars represent mean ± SEM. *, p<0.05; ***, p<0.001 compared with controls. #, p<0.05 compared with palmitate-treated cells.