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. 2014 May 9;289(19):13575-88.
doi: 10.1074/jbc.M113.531970. Epub 2014 Mar 27.

Increased Glucose Metabolism and Glycerolipid Formation by Fatty Acids and GPR40 Receptor Signaling Underlies the Fatty Acid Potentiation of Insulin Secretion

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Increased Glucose Metabolism and Glycerolipid Formation by Fatty Acids and GPR40 Receptor Signaling Underlies the Fatty Acid Potentiation of Insulin Secretion

Mahmoud El-Azzouny et al. J Biol Chem. .
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Abstract

Acute fatty acid (FA) exposure potentiates glucose-stimulated insulin secretion in β cells through metabolic and receptor-mediated effects. We assessed the effect of fatty acids on the dynamics of the metabolome in INS-1 cells following exposure to [U-(13)C]glucose to assess flux through metabolic pathways. Metabolite profiling showed a fatty acid-induced increase in long chain acyl-CoAs that were rapidly esterified with glucose-derived glycerol-3-phosphate to form lysophosphatidic acid, mono- and diacylglycerols, and other glycerolipids, some implicated in augmenting insulin secretion. Glucose utilization and glycolytic flux increased, along with a reduction in the NADH/NAD(+) ratio, presumably by an increase in conversion of dihydroxyacetone phosphate to glycerol-3-phosphate. The fatty acid-induced increase in glycolysis also resulted in increases in tricarboxylic cycle flux and oxygen consumption. Inhibition of fatty acid activation of FFAR1/GPR40 by an antagonist decreased glycerolipid formation, attenuated fatty acid increases in glucose oxidation, and increased mitochondrial FA flux, as evidenced by increased acylcarnitine levels. Conversely, FFAR1/GPR40 activation in the presence of low FA increased flux into glycerolipids and enhanced glucose oxidation. These results suggest that, by remodeling glucose and lipid metabolism, fatty acid significantly increases the formation of both lipid- and TCA cycle-derived intermediates that augment insulin secretion, increasing our understanding of mechanisms underlying β cell insulin secretion.

Keywords: Diabetes; Fatty Acid Metabolism; Fatty Acid Potentiation of GSIS; G Protein-coupled Receptors (GPCR); GPR40; Glucose Metabolism; Insulin Secretion; Metabolism; Metabolomics; β Cell.

Figures

FIGURE 1.
FIGURE 1.
Temporal metabolites profile of INS-1/832/3 cells upon glucose stimulation in the presence or absence of palmitic acid. The heat maps (center and right columns) are showing the metabolites levels expressed as fold change of the (−30 min) time point. INS-1 cells were incubated in RPMI with 3 mm glucose for 6 h (−30 time point) before incubation in KRHB for 30 min with no glucose in the presence or absence of 500 μm palmitate (0 time point). Cells were stimulated with 16.6 mm [12C]glucose for different time points (5–60 min). *, p < 0.05 in the peak areas, or their ratios, versus t = −30 min by analysis of variance using Tukey's post hoc analysis. The heat map in the left column shows the ratio of palmitate/BSA. *, p < 0.05 in the peak areas, or their ratios, between palmitate (Palm) or BSA at each time point by analysis of variance using Tukey's post hoc analysis. PPP, pentose phosphate pathway; LC-CoA, long chain-CoA; HILIC, lipids or phospholipids separated using hydrophilic interaction chromatography (HILIC).
FIGURE 2.
FIGURE 2.
Palmitic acid incubation effect on GSIS, AMPK, and related metabolites. A, insulin levels after stimulation with 16.6 mm glucose for different time intervals in the presence or absence of 500 μm palmitate. Also shown are levels of palmitoyl-CoA (B), palmitoyl-carnitine (C), malonyl-CoA (D), AMP (G), and ZMP (H) before and after 30-min incubation with 500 μm palmitate (time 0) and after stimulation with 16.6 mm glucose for different time points. A Western blot analysis for p-AMPK (E) and p-ACC (F) before glucose addition (time 0) and after 30 min of glucose stimulation (30 min) is shown. *, p < 0.05 between BSA and palmitate at each time point. Points represent mean ± S.E. n = 3–4/time point.
FIGURE 3.
FIGURE 3.
Palmitic acid effect on carnitines and glycerolipids metabolites. Glycerolipids levels before and after stimulation with 16.6 mm [U-13C]glucose with and without preincubation with 500 μm palmitate for 30 min (time 0) and after 5, 15, or 60 min of glucose stimulation (A–I and K). CDP-choline (J) and CDP-ethanolamine (CDP-eth) (L) are measured after stimulation with [12C]glucose. *, p < 0.05 between BSA and palmitate at each time point. Error bars represent mean ± S.E. n = 3–4.
FIGURE 4.
FIGURE 4.
Alterations in additional fatty acid identified by untargeted metabolomic profiling and effects on insulin secretion. A–C, levels of palmitoyl glycine, palmitoyl taurine, and sphingosine phosphate after incubation with fatty acid for 30 min, followed by stimulation with 16.6 mm [U-13C]glucose for 5,15, or 60 min. D and E, levels of palmitoyl taurine and palmitoyl glycine after the addition of increasing concentrations of their precursor amino acids in the presence or absence of palmitate. F, insulin levels after incubation of cells with BSA or palmitic acid in the presence or absence of glycine. Medium was collected before addition of glucose (0 min) or after stimulation with 16.6 mm glucose for 20 or 60 min. Error bars represent mean ± S.E. n = 3–4. *, p < 0.05 between BSA and palmitate at each time point unless described otherwise on the graph.
FIGURE 5.
FIGURE 5.
Palmitic acid effect on glycolysis, pentose phosphate metabolites, and the TCA cycle. Shown are changes in levels of fructose bisphosphate (A) phosphogluconate (C), ribose and ribulose-P (E), glycerol-3-phosphpate (G), 2PG + 3PG (I), citrate (J), malate (K), and NADH (L) after stimulation with 16.6 mm [12C]glucose in the presence or absence of 500 μm palmitate. Also shown are changes in total mass and 13C isotopologues after stimulation with 16.6 mm [U-13C]glucose for fructose bisphosphate (B), phosphogluconate (D), ribose and ribulose-P (F), and glycerol-3-phosphate (H). Error bars represent mean ± S.E. n = 3–4, except for control at 60 min using [U-13C]glucose, where n = 2. The values were confirmed with n = 4 in a separate experiment. *, p < 0.05 between BSA and palmitate at each time point.
FIGURE 6.
FIGURE 6.
Oleate and palmitate effect on different metabolites. INS-1 832/13 cells were incubated in RPMI medium with low glucose for 6 h before incubation with either 500 μm palmitate, 500 μm oleate, or 0.5% BSA for 30 min. Preincubation was followed by stimulation with 16.6 mm [U-13C]glucose for 60 min. Changes in the 13C isotopologues are shown for glycerol-3-phosphate (A), 2PG + 3PG (B), NADH/NAD+ (C), malonyl-CoA (D), long chain CoA (E), lysophosphatidic acid (F), diacylglycerol (G), and triglycerides (H). The relative peak area ± S.E. of the indicated metabolites was assessed. n = 3–4 for each metabolite.
FIGURE 7.
FIGURE 7.
Palmitic acid effect on glycolytic flux, fatty acid oxidation, oxygen consumption, and glucose utilization. A, for the pulse-chase experiment, cells were stimulated with 16.7 mm [U-13C]glucose for 15 min (pulse) before the KRHB was replaced with the same medium containing 16.7 mm [12C]glucose for 2 min, after which the cells were quenched and the rate of consumption of labeled Go3P and the rate of formation of unlabeled Go3P in 2 min was plotted. B and C, the OCR and extracellular acidification rate were measured before and after the addition of 16.6 mm glucose. D, the OCR of cells incubated with 500 μm palmitate before the addition of 0.2 mm etomoxir followed by 16.7 mm glucose stimulation. E, the percentage labeling of citrate and isocitrate (IsoCit) after incubation of INS-1 cells with either BSA or [U-13C]palmitate or [U-13C]oleate for 30 min with no glucose (time 0) and after stimulation with 16.6 mm [12C]glucose for 60 min (time 60). Citr, citrate. F, the percentage labeling of ultimate labeling of citrate, aspartate, and malate after 1-h treatment with [U-13C]glucose. G and H, glucose utilization using 5-[3H]glucose in the presence or absence of 100 or 500 μm palmitate. Error bars represent mean ± S.E. with n = 3–4 for metabolites analysis and n = 10 for SeaHorse experiments and n = 3–10 for glucose utilization. *, p < 0.05 between BSA and palmitate at each time point.
FIGURE 8.
FIGURE 8.
GPR40 role in palmitic acid-induced metabolic changes. INS-1 cells were incubated with 250 μm palmitate or 0.25% BSA in the presence or absence of 5 μm GPR40 antagonist (GW1100) for 30 min, followed by stimulation with 16.6 mm [U-13C]glucose for 60 min. Using these conditions, the following were measured: insulin levels (A), changes in total mass and 13C isotopologues of glycerol-3-phosphate (B), hexose phosphates (C), LPA (16:0) and DAG(32:0) (E), DAG (34:1) (F), and CDP-ethanolamine (L). INS-1 cells were incubated with 50 μm palmitate or BSA in the presence or absence of 10 μm Cay 10587 for 30 min, followed by stimulation with 16.6 mm [U-13C]glucose for 60 min. G, accumulation of DAG (34:1). INS-1 cells were incubated with 50 μm palmitate or BSA in the presence or absence of 5 μm TAK 875 for 30 min, followed by stimulation with 16.6 mm [U-13C]glucose for 30 min. Under these conditions, the following were measured: fructose bisphosphate (FBP) (I), NADH/NAD+ ratio (J), DAG (32:0) (K), and CDP-ethanolamine (L). Error bars represent mean ± S.E. n = 3–4.
FIGURE 9.
FIGURE 9.
Metabolic changes with GPR40 modulation. INS-1 cells were incubated with 250 μm palmitate ± 5 μm GW1100 in the absence of glucose for 30 min, and the levels of palmitoyl-carnitine (A) and palmitoyl-CoA (C) were measured at different time points. INS-1 cells were incubated with 50 μm palmitate ± 10 μm Cay 10587 GPR40 agonist in the absence of glucose for 30 min, and the levels of palmitoyl-carnitine (B) and palmitoyl-CoA (D) were measured at different time points. I–L, INS-1 cells were incubated with 50 μm palmitate in the presence or absence of 5 μm TAK 875. E, INS-1 cells were incubated with 50 μm [U-13C]palmitate (Palm) in the absence of glucose ± Cay 10587 agonist for 30 min, and the ratio of labeled DAG (34:1)+16 to unlabeled DAG (34:1) was measured. F, INS-1 cells were incubated with 50 μm [U-13C]palmitate in the absence of glucose ± TAK 875 for 30 min before stimulation with [12C]glucose for another 30 min, and the change in total mass and 13C isotopologues of labeled DAG (34:1) was measured. Shown is the oxygen consumption rate in the presence or absence of agonist (Cay10587) (G) or antagonist (GW1100) (H) before and after stimulation with 16.6 mm glucose. Error bars represent mean ± S.E. n = 3 or 4 for metabolite analysis and n = 6–7 for the SeaHorse experiment. *, p < 0.05.
FIGURE 10.
FIGURE 10.
Proposed pathways for fatty acid metabolism during the fed and fasting state and its role in potentiating insulin secretion. In β cells, fatty acids increase glucose flux to glycerol-3-phosphate (Glycerol-3-P) to increase formation of lysophosphatidic acid (LPA) and downstream signaling molecules. The increased conversion of dihydroxyacetone phosphate regenerates NAD+, which increases glycolytic flux and increases TCA cycle activity. FFAR1/GPR40 receptor activation appears to enhance the formation of LPA and increase glycerolipid cycling. LC-CoA, long chain CoA.

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