Free fatty acids (FFAs) have pleiotropic effects on the pancreatic β-cell. Although acute exposure to FFAs stimulates glucose-stimulated insulin secretion (GSIS), prolonged exposure impairs GSIS and causes apoptosis. FFAs exert their effects both via intracellular metabolism and interaction with the FFA receptor 1 (FFAR1/GPR40). Here we studied the role of FFAR1 in acute and long-term effects of palmitate on GSIS and insulin content in isolated human islets by using the FFAR1 agonist TAK-875 and the antagonist ANT203. Acute palmitate exposure potentiated GSIS approximately 3-fold, whereas addition of the antagonist decreased this potentiation to approximately 2-fold. In the absence of palmitate, the agonist caused a 40% increase in GSIS. Treatment with palmitate for 7 days decreased GSIS to 70% and insulin content to 25% of control level. These negative effects of long-term exposure to palmitate were ameliorated by FFAR1 inhibition and further aggravated by additional stimulation of the receptor. In the absence of extracellularly applied palmitate, long-term treatment with the agonist caused a modest increase in GSIS. The protective effect of FFAR1 inhibition was verified by using FFAR1-deficient MIN6 cells. Improved β-cell function by the antagonist was paralleled by the decreased apoptosis and lowered oxidation of palmitate, which may represent the potential mechanisms of protection. We conclude that FFAR1 in the pancreatic β-cell plays a substantial role not only in acute potentiation of GSIS by palmitate but also in the negative long-term effects of palmitate on GSIS and insulin content.

Folding and post-translational modification of the thyroid hormone precursor, thyroglobulin (Tg), in the endoplasmic reticulum (ER) of the thyroid epithelial cells is facilitated by several molecular chaperones and folding enzymes, such as BiP, GRP94, calnexin, protein disulfide isomerase, ERp72, and others. They have been shown to associate simultaneously and/or sequentially with Tg in the course of its maturation, thus forming large heterocomplexes in the ER of thyrocytes. Here we present evidence that such complexes include a novel member, an ER-resident lumenal protein, ERp29, which is present in all mammalian tissues with exceptionally high levels of expression in the secretory cells. ERp29 was induced upon treatment of FRTL-5 rat thyrocytes with the thyroid-stimulating hormone, which is essential for the maintenance of thyroid cells and Tg biosynthesis. Chemical cross-linking followed by the cell lysis and immunoprecipitation of ERp29 or Tg revealed association of these proteins and additionally, immunocomplexes that also included major ER chaperones, BiP and GRP94. Sucrose density gradient analysis indicated colocalization of ERp29 with Tg and BiP in the fractions containing large macromolecular complexes. This was supported by immunofluorescent microscopy showing co-localization of ERp29 with Tg in the putative transport vesicular structures. Affinity chromatography using Tg as an affinity ligand demonstrated that ERp29 might be selectively isolated from the FRTL-5 cell lysate or purified lumenal fraction of rat liver microsomes along with the other ER chaperones. Preferential association with the urea-denatured Tg-Sepharose was indicative of either direct or circuitous ERp29/Tg interactions in a chaperone-like manner. Despite the presence of the C-terminal ER-retrieval signal, significant amounts of ERp29 were also recovered from the culture medium of stimulated thyrocytes, indicating ERp29 secretion. Based on these data, we suggest that the function of ERp29 in thyroid cells is connected with folding and/or secretion of Tg.

BackgroundFree fatty acids (FFAs) acutely stimulate insulin secretion from pancreatic islets. Conflicting results have been presented regarding this effect at non-stimulatory glucose concentration, however. The aim of our study was to investigate how long-chain FFAs affect insulin secretion from isolated human pancreatic islets in the presence of physiologically fasting glucose concentrations and to explore the contribution of mitochondria to the effects on secretion.MethodsInsulin secretion from human pancreatic islets was measured from short-term static incubation or perfusion system at fasting glucose concentration (5.5 mM) with or without 4 different FFAs (palmitate, palmitoleate, stearate, and oleate). The contribution of mitochondrial metabolism to the effects of fatty acid-stimulated insulin secretion was explored.ResultsThe average increase in insulin secretion, measured from statically incubated and dynamically perifused human islets, was about 2-fold for saturated free fatty acids (SFAs) (palmitate and stearate) and 3-fold for mono-unsaturated free fatty acids (MUFAs) (palmitoleate and oleate) compared with 5.5 mmol/l glucose alone. Accordingly, MUFAs induced 50 % and SFAs 20 % higher levels of oxygen consumption compared with islets exposed to 5.5 mmol/l glucose alone. The effect was due to increased glycolysis. When glucose was omitted from the medium, addition of the FFAs did not affect oxygen consumption. However, the FFAs still stimulated insulin secretion from the islets although secretion was more than halved. The mitochondria-independent action was via fatty acid metabolism and FFAR1/GPR40 signaling.ConclusionsThe findings suggest that long-chain FFAs acutely induce insulin secretion from human islets at physiologically fasting glucose concentrations, with MUFAs being more potent than SFAs, and that this effect is associated with increased glycolytic flux and mitochondrial respiration.

Prolonged exposure to palmitate impairs β-cell function and mass. One of the proposed mechanisms is alteration in ceramide (Cer) generation. In the present study, exposure to palmitate induced the level of palmitoyl transferase and Cer synthases, enzymes of the Cer de novo and salvage pathways, and doubled total Cer levels, which was associated with decreased insulin secretion and augmented apoptosis in MIN6 cells and human islets. By inhibiting enzymes of the pathways pharmacologically with myriocin (ISP-1) or fumonisin B1 or by small interfering RNA (siRNA), we showed that Cer(14:0), Cer(16:0), Cer(20:1), and Cer(24:0) species, generated by the salvage pathway, are linked to the harmful effect of palmitate on β-cells. Oleate attenuates negative effects of palmitate on β-cells. When oleate was included during culture of MIN6 cells with palmitate, the palmitate-induced up-regulation of the enzymes of the de novo and salvage pathways was prevented resulting in normalized levels of all Cer species except Cer(20:1). Our data suggest that enhanced Cer generation in response to elevated palmitate levels involves both de novo and salvage pathways. However, the negative effects of palmitate on β-cells are attributed to generation of Cer species Cer(14:0), Cer(16:0), and Cer(24:0) via acylation of sphingosine.

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