Chronic Exposure to Palmitate Impairs Insulin Signaling in an Intestinal L-cell Line: A Possible Shift from GLP-1 to Glucagon Production

Abstract

Obesity and type 2 diabetes mellitus (T2DM) are characterized by insulin resistance and impaired glucagon-like peptide-1 (GLP-1) secretion/function. Lipotoxicity, a chronic elevation of free fatty acids in the blood, could affect insulin-signaling in many peripheral tissues. To date, the effects of lipotoxicity on the insulin receptor and insulin resistance in the intestinal L-cells need to be elucidated. Moreover, recent observations indicate that L-cells may be able to process not only GLP-1 but also glucagon from proglucagon. The aim of this study was to investigate the effects of chronic palmitate exposure on insulin pathways, GLP-1 secretion and glucagon synthesis in the GLUTag L-cell line. Cells were cultured in the presence/absence of palmitate (0.5 mM) for 24 h to mimic lipotoxicity. Palmitate treatment affected insulin-stimulated GLP-1 secretion, insulin receptor phosphorylation and IRS-1-AKT pathway signaling. In our model lipotoxicity induced extracellular signal-regulated kinase (ERK 44/42) activation both in insulin stimulated and basal conditions and also up-regulated paired box 6 (PAX6) and proglucagon expression (Gcg). Interestingly, palmitate treatment caused an increased glucagon secretion through the up-regulation of prohormone convertase 2. These results indicate that a state of insulin resistance could be responsible for secretory alterations in L-cells through the impairment of insulin-signaling pathways. Our data support the hypothesis that lipotoxicity might contribute to L-cell deregulation.

Keywords: insulin resistance; intestinal L-cells; lipotoxicity; proglucagon.

Figure 1

Effect of pre-exposure to palmitate on cell viability and lipid accumulation in GLUTag cells. A: MTT assay in GLUTag cells pre-exposed to palmitate (0.25, 0.50 and 1.00) after 12 h, 24 h and 48 h. Data are expressed as means ± standard error of 570 nM absorbance to % of control. * p < 0.05, ** p < 0.01, vs. control (t-test, n = 6). B: Nile Red staining in GLUTag cells pre-exposed to palmitate (0.25, 0.50 and 1.00 for 24 h). Data are expressed as means ± standard error of fluorescence to % of control. * p < 0.05, ** p < 0.01, vs. control (t-test, n = 6). C: Oil red O staining in GLUTag cells treated with palmitate (0.25, 0.50 and 1.00 for 24 h). A slight increase in Oil red O stained droplets (red) is visible in the cells treated with palmitate (0.50 and 1.00 mM) as compared with non-treated cells (40× magnification).

Figure 2

Effect of pre-exposure to palmitate on glucagon-like peptide-1 (GLP-1) secretion in GLUTag cells. Acute insulin-induced GLP-1 secretion in control cells (open bars) and in cells pre-exposed to 0.5 mM of palmitate for 24 h (gray bars). * p < 0.05, *** p < 0.001 vs. basal level in control group; ^### p < 0.001 vs. insulin stimulated control group, n.s. not significant (1-way ANOVA followed by Bonferroni test, n = 4); (+) means presence, (-) means absence.

Figure 3

Effect of pre-exposure to palmitate on IR phosphorylation and the IRS-1/AKT pathway in GLUTag cells. Representative immunoblot from control and palmitate GLUTag treated cells (0.5 mM for 24 h) acutely stimulated with insulin 10⁻⁹ M for 5 min for: A: immunoprecipitation of the total IR (Tyr1150/1151 on the β subunit) (p-IR-β) and total IR; B: p-IRS-1 (Tyr612) and total IRS-1 (IRS-1); C: p-AKT (Ser 473) and total AKT (AKT); D: corresponding densitometric analysis in control cells (open bars) and in cells exposed to palmitate (gray bars). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. basal control; ^## p < 0.01, ^### p < 0.001 vs. insulin stimulated control group; ^§§ p < 0.01 vs. palmitate; n.s. not significant (1-way ANOVA followed by Bonferroni test, n = 3); (+) means presence, (-) means absence.

Figure 4

Effect of pre-exposure to palmitate on MAPK pathway activation in GLUTag cells. A: representative Western blot for p-ERK 44/42, total ERK 44/42 (ERK) and β-actin from control and palmitate GLUTag treated cells (0.5 mM for 24 h) acutely stimulated with insulin 10⁻⁹ M for 5 min. B: corresponding densitometric analysis in control cells (open bars) and in cells exposed to palmitate (gray bars). *** p < 0.001 vs. basal control; ^### p < 0.001 vs. insulin stimulated control group; n.s. not significant (1-way ANOVA followed by Bonferroni test, n = 3); (+) means presence, (-) means absence.

Figure 5

IR-A and IR-B mRNA expression in GLUTag cells. A: representative agarose gel image for RT-PCR analysis of IR-B (286 bp) and IR-A (250 bp) mRNA expression in control and palmitate treated cells (0.5 mM for 24 h). “-“ represents PCR reaction without added template. B: statistical data showing the mRNA ratio of IR-A/IR-B in control cells (open bars) and in cells exposed to palmitate (gray bars) (n = 3, *** p < 0.001 compared to control); (+) means presence, (-) means absence.

Figure 6

Effect of palmitate on PAX6 and proglucagon expression in GLUTag cells. A: representative Western blot for PAX6 and β-actin from control and palmitate GLUTag treated cells (0.5 mM for 24 h). B: corresponding densitometric analysis in control cells (open bars) and in cells exposed to palmitate (gray bars). *** p < 0.001 vs. control (1-way ANOVA followed by Bonferroni test, n = 3). C: box plot of proglucagon (GcG) mRNA expression in control and palmitate treated cells (0.5 mM for 24 h). Values on the y-axis are reported as 2^−ΔΔCt. Statistical significance was evaluated by t-test (n = 3, * p < 0.05 compared to control); (+) means presence, (-) means absence.

Figure 7

Effects of pre-exposure to palmitate on PC2 protein expression and glucagon secretion. A: representative Western blot for PC2 and β-actin from control and palmitate GLUTag treated cells (0.5 mM for 24 h). B: corresponding densitometric analysis in control cells (open bars) and in cells exposed to palmitate (gray bars). * p < 0.05 vs. control (1-way ANOVA followed by Bonferroni test, n = 3). C: glucagon secretion in control cells (open bars) and in cells pre-exposed to 0.5 mM of palmitate for 24 h (gray bars). * p < 0.05 vs. basal level in the control group; ^### p < 0.001 vs. insulin stimulated control group; § p < 0.05 vs basal level in the palmitate group (1-way ANOVA followed by Bonferroni test, n = 4); (+) means presence, (-) means absence.

Figure 8

Effect of MAPK pathway inhibition on proglucagon gene expression in GLUTag cells pre-exposed to palmitate. A: representative Western blot for p-ERK 44/42, ERK 44/42 and β-actin from control and palmitate GLUTag treated cells (0.5 mM for 24 h) in the absence or presence of the U0126 (MAPK Inhibitor). B: corresponding densitometric analysis in control cells (open bars) and in cells exposed to palmitate (gray bars). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control; ^§§§ p < 0.001 vs. palmitate (1-way ANOVA followed by Bonferroni test, n = 3). C: Box plot of proglucagon (GcG) mRNA expression in control cells (open bars) and in 0.5 mM palmitate treated cells for 24 h (gray bars) in the absence or presence of U0126. Values on the y-axis are reported as 2^−ΔΔCt. Statistical significance was evaluated by t-test (n = 3, * p < 0.05, *** p < 0.001 vs. control); (+) means presence, (-) means absence.

Figure 9

Effects of MAPK pathway inhibition on PAX6 and PC2 protein expression in GLUTag cells pre-exposed to palmitate. Representative Western blot from control and palmitate GLUTag treated cells (0.5 mM for 24 h) in the absence or presence of U0126 (MAPK Inhibitor) for: A: PAX6 and β-actin; B: PC2 and β-actin; C: corresponding densitometric analysis in control cells (open bars) and in cells exposed to palmitate (gray bars). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control; § p < 0.05, ^§§§ p < 0.001 vs. palmitate (1-way ANOVA followed by Bonferroni test, n = 3); (+) means presence, (-) means absence.