Palmitic acid-induced lipotoxicity promotes a novel interplay between Akt-mTOR, IRS-1, and FFAR1 signaling in pancreatic β-cells

Abstract

Background: Free fatty acid receptor 1 (FFAR1) is G-protein coupled receptor predominantly expressed in pancreatic β-cells that is activated by a variety of free fatty acids (FFAs). Once activated, it promotes glucose-stimulated insulin secretion (GSIS). However, increased levels of FFAs lead to lipotoxicity, inducing loss of β-cell function. FFAR1 plays a key role in the development of type 2 diabetes (T2D), and previous studies have indicated the importance of developing anti-diabetic therapies against FFAR1, although its role in the regulation of β-cell function remains unclear. The present study investigated the role of FFAR1 under lipotoxic conditions using palmitic acid (PA). The rat insulinoma 1 clone 832/13 (INS-1 832/13) cell line was used as a model as it physiologically resembles native pancreatic β-cells. Key players of the insulin signaling pathway, such as mTOR, Akt, IRS-1, and the insulin receptor (INSR1β), were selected as candidates to be analyzed under lipotoxic conditions.

Results: We revealed that PA-induced lipotoxicity affected GSIS in INS-1 cells and negatively modulated the activity of both IRS-1 and Akt. Reduced phosphorylation of both IRS-1 S636/639 and Akt S473 was observed, in addition to decreased expression of both INSR1β and FFAR1. Moreover, transient knockdown of FFAR1 led to a reduction in IRS-1 mRNA expression and an increase in INSR1β mRNA. Finally, PA affected localization of FFAR1 from the cytoplasm to the perinucleus.

Conclusions: In conclusion, our study suggests a novel regulatory involvement of FFAR1 in crosstalk with mTOR-Akt and IRS-1 signaling in β-cells under lipotoxic conditions.

Keywords: Akt; FFAR1; INSR; IRS-1; Insulin resistance; Lipotoxicity; Palmitic acid; Type 2 diabetes; mTOR; β-cells.

Fig. 1

Effect of PA-induced lipotoxicity on INS-1 cells. INS-1 cells were treated with various concentrations of PA for 24 h followed by MTT cell survival assay. Triplicate cell repeats were performed for each group. The absorbance at 570 nm was measured and showed a decrease in the percentage of cell survival with increasing doses of PA compared to the vehicle control. LD₅₀ was calculated as 0.4 mM PA (indicated by the dotted lines). Values represent mean ± SEM and the data are representative of at least three independent experiments

Fig. 2

Effect of high PA concentrations on GSIS in INS-1 cells. a GSIS was performed using the perifusion method in which triplicate repeats of 300 clusters of INS-1 cells were pretreated with PA for 24 h and then exposed to different glucose concentrations at 5- and 15-min time points. A gradual decrease in GSIS was observed between the control and the PA-treated groups, at both low and high glucose concentrations. b However, the difference in both the first (5 min) and second (15 min) phase of insulin secretion was more prominent when cells were exposed to increased lipotoxic conditions (0.4 and 0.6 mM PA) indicated by their stimulation indexes. c A similar pattern in GSIS was observed when monolayer INS-1 cells were exposed for a longer period to different glucose concentrations using the static incubation method. Data represent mean ± SEM of normalized insulin/DNA of at least three independent experiments. *P < 0.05 and **P < 0.01

Fig. 3

Effect of PA on protein expression in INS-1 cells. Cells were grown in a monolayer culture and treated for 24 h with different concentrations of PA. Duplicate cell repeats were performed for each group. Whole-cell lysates were separated by 8–12% SDS-PAGE and then analyzed by Western blotting. a The left panel illustrates both mTOR and insulin signaling pathway targets and shows a reduction in expression levels with treatment using 0.4 and 0.6 mM PA compared with the control. The right panel shows there were no significant changes in target expression with PA treatment. Actin was used as a loading control. b Protein quantification of targets affected by PA in (a) and their statistical significance are shown. Values represent mean ± SEM and the data are representative of at least three independent experiments. *P < 0.05 and **P < 0.01

Fig. 4

PA promotes FFAR1 mRNA expression in INS-1 cells. Cells were grown in a monolayer culture and treated with different concentrations of PA for 24 h; then, RNA was extracted and analyzed by qPCR. Duplicate cell repeats were performed for each group. No significant changes were seen in mRNA levels for both INSR1β and GLUT2 following treatment with 0.4 or 0.6 mM PA compared with the untreated controls. However, there was a clear increase in FFAR1 levels following exposure to 0.4 and 0.6 mM PA. GAPDH was used as a reference gene for normalization. Values represent mean ± SEM and the data are representative of at least three independent experiments. *P < 0.05 and **P < 0.01

Fig. 5

Effect of FFAR1 knockdown on mTOR and insulin signaling in INS-1 cells. Cells were transiently transfected with either 40 nM scrambled siRNA or FFAR1 siRNA for 48–72 h, harvested, and analyzed by qPCR. Duplicate cell repeats were performed for each group. Transfections yielded an efficiency of 75% (data not shown). a Around 60% FFAR1 knockdown was achieved on the mRNA level and led to a significant decrease in IRS-1 and an increase in INSR1β mRNA expression. Other targets of the mTOR and insulin signaling, such as PI3k, Raptor, Rictor, and S6K1, were not affected. GAPDH was used as a reference gene for normalization. b A representative Western blot of the protein expression and quantification is shown where approximately 30% FFAR1 knockdown was achieved that had a significant downregulatory effect on IRS-1 protein levels, but not on INSR1β. Values represent mean ± SEM and the data are representative of at least three independent experiments. *P < 0.05 and **P < 0.01

Fig. 6

PA causes cytoplasmic to perinuclear translocation of FFAR1 in INS-1 cells. Cells were treated with 0.6 mM PA for 24 h, fixed and stained, and analyzed by confocal microscopy using a ×100 objective lens. Additionally, cells were treated with 0.6 mM PA for 24 h and then re-incubation for another 24 h with complete media. Duplicate cell repeats were performed for each group. FFAR1 conjugated with Alexa Fluor 594 (red) translocated from the cytoplasm to the perinucleus following PA treatment (middle panel), but not in the untreated cells (top panel). However, FFAR1 remained in the perinucleus despite the reintroduction of complete media (bottom panel). The mean intensity of FFAR1 for each condition was measured by ZEISS ZEN 2010 ver. 6.0 software as follows; untreated (2.9), 24 h PA (4.0), and 24 h PA + CM (4.29). Nuclei were stained using DAPI (blue). The shown data are representative of at least three independent experiments. CM complete media

Fig. 7

Proposed model of the crosstalk between mTORC2, IRS-1, and FFAR1 signaing in β-cell under lipotoxic conditions. In the absence of PA, FFAR1 is predominantly located at the cell surface and/or cytoplasm due to the absence of PTMs allowing it to exert its effect indirectly (dashed arrow) on mTORC2, and consequently, phosphorylating and activating Akt at S473 (left panel). On the other hand, PA-induced lipotoxicity mobilizes FFAR1 to perinuclear structures due to possible PTMs attenuating the activity of mTORC2 (dashed line). This also has an indirect inhibitory effect (dashed line) on the double phosphorylation of IRS-1 at S636/639 (right panel). P phosphorylation, PTM posttranslational modification