1986 Diabetes Volume 67, October 2018 Endogenous Fatty Acids Are Essential Signaling Factors of Pancreatic b-Cells and Insulin Secretion Sebastian Hauke,1 Kaya Keutler,1,2 Prasad Phapale,1 Dmytro A. Yushchenko,1,3 and Carsten Schultz1,2 Diabetes 2018;67:1986–1998 | https://doi.org/10.2337/db17-1215 The secretion of insulin from b-cells depends on extra- insulin is required to sustain normoglycemia compared cellular factors, in particular glucose and other small with a constant insulin supply (2). Disturbed synchronized molecules, some of which act on G-protein–coupled oscillatory insulin secretion was found in hyperglycemic receptors. Fatty acids (FAs) have been discussed as and hyperinsulinemic ob/ob mice and was suggested to exogenous secretagogues of insulin for decades, espe- contribute to insulin resistance and to the development of cially after the FA receptor GPR40 (G-protein–coupled type 2 diabetes mellitus (3). The coordination of islets in receptor 40) was discovered. However, the role of FAs the pancreas, in addition to gap junctions (4), relies on as endogenous signaling factors has not been investi- indirect intercellular communication (i.e., on soluble mes- gated until now. In the present work, we demonstrate sengers that reach b-cells by diffusion in the intercellular that lowering endogenous FA levels in b-cell medium by space of islets and across pancreatic lobes) (5). Individual stringent washing or by the application of FA-free (FAF) b-cell activity as well as cell-to-cell coordination in pan- BSA immediately reduced glucose-induced oscillations 2+ 2+ creatic islets are predominantly fine-tuned by the close of cytosolic Ca ([Ca ]i oscillations) in MIN6 cells and mouse primary b-cells, as well as insulin secretion. Mass coordination of a multitude of diffusible factors that os- spectrometry confirmed BSA-mediated removal of FAs, cillate in the extracellular space. This includes a number of with palmitic, stearic, oleic, and elaidic acid being the most neurotransmitters and hormones, such as g-aminobutyric abundant species. [Ca2+] oscillations in MIN6 cells recov- acid (6) and neuropeptide Y (7). In particular, b-cells are i – ered when BSA was replaced by buffer or as FA levels in the equipped with many G-protein coupled receptors, which SIGNAL TRANSDUCTION 2+ supernatant were restored. This was achieved by recombi- are involved in intracellular Ca signaling and significantly nant lipase–mediated FA liberation from membrane lipids, contribute to orchestrating oscillations of the intracellular 2+ 2+ by the addition of FA-preloaded FAF-BSA, or by the photol- free Ca concentration ([Ca ]i oscillations) and insulin ysis of cell-impermeant caged FAs. Our combined data secretion. Examples are purinergic receptors that are trig- support the hypothesis of FAs as essential endogenous gered by the auto- and paracrine release of ATP from b-cells signaling factors for b-cell activity and insulin secretion. (8). In 2003, three independent groups reported that GPR40, a G-protein–coupled receptor that is abundantly expressed in the brain and in the pancreas, acts as a recep- In vertebrates, insulin secretion is a synchronized multi- tor for medium- and long-chain fatty acids (FAs) (9–11). cellular process. Sufficiently high blood insulin levels are GPR40 was found to mediate the majority of the effects ensured by the coordinated stimulation of b-cells in pan- that FAs cause on insulin secretion, promoting increased 2+ creatic islets. Coupling of the secretory activity of pancre- [Ca ]i (12,13). Deletion of GPR40 expression in pancre- atic islets results in oscillatory elevations of insulin levels atic b-cells led to resistance toward this FA-induced re- 2+ in the bloodstream (1). Studies on patients with diabetes sponse in [Ca ]i oscillations and insulin secretion (12,14). indicate that under oscillatory infusion of insulin, less GPR40 overexpression, on the other hand, was described to 1Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, This article contains Supplementary Data online at http://diabetes Heidelberg, Germany .diabetesjournals.org/lookup/suppl/doi:10.2337/db17-1215/-/DC1. 2 Department of Physiology and Pharmacology, Oregon Health & Science Univer- © 2018 by the American Diabetes Association. Readers may use this article as sity, Portland, OR long as the work is properly cited, the use is educational and not for profit, and the 3 Institute of Organic Chemistry and Biochemistry, Academy of Sciences of Czech work is not altered. More information is available at http://www.diabetesjournals Republic, Prague, Czech Republic .org/content/license. Corresponding authors: Carsten Schultz, [email protected], and Dmytro A. See accompanying article, p. 1932. Yushchenko, [email protected]. Received 6 October 2017 and accepted 2 May 2018. diabetes.diabetesjournals.org Hauke and Associates 1987 augment glucose-stimulated insulin secretion (GSIS) (15), Isolation and Culture of Mouse Primary b-Cells 2+ pointing to a major role of GPR40 in FA-stimulated [Ca ]i Mouse islets were isolated from C57BL/6 mice (female signaling and GSIS. Recently, we have shown that photo- Ctrl:CD1(ICR), outbred (catalog #CD1S1FE07W; Charles release of arachidonic acid leads to GPR40 stimulation River Laboratories) by collagenase digestion, as previously and subsequent activation of trimeric G-proteins together described (25). Briefly, mice were euthanized by cervical 2+ with [Ca ]i signaling in MIN6 cells (16). For decades, dislocation, and a collagenase solution (1 mg/mL) was elevated levels of FAs in the bloodstream have been de- injected into the bile duct, followed by digestion at 37°C scribed to significantly augment GSIS in isolated islets (17), for 10 min and separation of islets using a Histopaque animals (18), and nonobese human subjects (19). Studies in (Sigma-Aldrich) gradient (1.083 and 1.077 g/mL). Islets rat islets indicate a hypersensitivity of b-cells to glucose were cultured in RPMI medium (supplemented with 10% after prolonged exposure to FAs (20). In turn, clinical FCS, 100 units/mL penicillin, and 100 mg/mL strepto- studies correlated artificial decreases of plasma FA levels mycin) for 24 h. Upon trypsin digestion (5 min at 37°C), with the reduction of insulin secretion, indicating an es- islets were dissociated into single b-cells, which were sential role of circulating FAs for efficient GSIS (21,22). seeded on poly-L-lysine–coated coverslips. Animals were Even though FAs have been discussed as insulin secreta- housed in the European Molecular Biology Laboratory gogues for decades (as reviewed in Nolan et al. [23]), the role animal facilities under veterinarian supervision and the of endogenous FAs in single b-cell activity, intercellular and guidelines of the European Commission (revised directive interislet signaling, and in the synchronization of b-cells 2010/63/EU, and American Veterinary Medical Association has not been investigated so far. In the current study we guidelines 2007). therefore aimed 1) to determine the role of endogenous FAs Mouse Insulin ELISA as essential endogenous signaling factors for b-cell activity ELISA for the specific quantification of insulin was per- and insulin secretion and 2) to examine whether b-cell formed in 96-well format (Mercodia AB, Uppsala, Sweden). activity can be modulated by the removal or replenishment Insulin levels were normalized to the protein content of of endogenous FAs. Our approach to subordinate b-cell MIN6 cells, using a BCA assay (Pierce BCA Protein assay activity and insulin secretion to the presence of endogenous kit; Thermo Fisher Scientific). Experiments for the de- signaling factors, in particular FAs, contributes to a more termination of supernatant (SN) insulin content were detailed and complete understanding of the fundamental performed in quadruplicate per condition. regulation of b-cell activity and insulin secretion. Confocal Laser Scanning Microscopy RESEARCH DESIGN AND METHODS Imaging was performed on a FluoView1200 (catalog #IX83; Olympus) confocal laser-scanning microscope at 37°C (in- Reagents cubator box made by the European Molecular Biology Lab- BSA (FA free [FAF]: catalog #A7030, lot #SLBK9735V; oratory), using Olympus 603 Plan-Apo (numerical aperture conventional: catalog #A7906, lot #SLBR0420V), 13C-oleic, 1.4, oil) or 203 UPLSAPO (numerical aperture 0.75, air) 13C-palmitic, and 13C-decanoic acid were obtained from Sigma- objectives and FluoView software, version 4.2. The images Aldrich (St. Louis, MO). Thermo Fisher Scientific(Carlsbad, were acquired with a Hamamatsu C9100-50 EM-CCD cam- CA) provided gradient-grade Gibco FBS (catalog #16000–044) era. The green channel was imaged using a 488-nm laser and DMEM. Solvents were obtained in the highest analytical 2 line (120 mW/cm , 2.5%) and a 525/50 emission mirror. The grade from Biosolve (Valkenswaard, the Netherlands) and red channel was imaged using a 559-nm laser (120 mW/cm2, Sigma-Aldrich. All other commercially available chemicals 2.0%) and a 643/50 emission filter. Images were acquired and enzymes were purchased from Sigma-Aldrich and Cay- in 4-s intervals (frame time 3.9 s). A pulsed 375-nm laser man Chemical (Ann Arbor, MI). See Supplementary Data for fi (10 MHz) was applied for uncaging experiments in the en- unit de nitions of applied recombinant enzymes. tire field of view for eight frames (3.2 s/frame). The dual scanner setup allowed for simultaneous laser stimulation and Tissue Culture confocal imaging. This permitted the acquisition of cellular – Insulin-secreting MIN6 cells (24) (passage 25 36) were responses that occur during or immediately after laser stim- cultured at 37°C and 8% CO2 in a humidified atmosphere. 2+ ulation. To test changes in [Ca ]i at the single-cell level, MIN6 cells were grown in DMEM containing 4.5 g/L MIN6 cells, grown to 70% confluence in pseudoislets, were glucose, supplemented with 15% FBS and 70 mmol/L incubated with an acetoxymethyl ester of the Ca2+ indicator b-mercaptoethanol (PAN Biotech, Aidenbach, Germany).