ORIGINAL ARTICLE Glucose- and Hormone-Induced cAMP Oscillations in a- and b-Cells Within Intact Pancreatic Islets Geng Tian, Stellan Sandler, Erik Gylfe, and Anders Tengholm OBJECTIVE—cAMP is a critical messenger for insulin and and by stimulating mobilization and priming of granules via glucagon secretion from pancreatic b- and a-cells, respectively. protein kinase A– and Epac-dependent pathways (7,8). Dispersed b-cells show cAMP oscillations, but the signaling ki- Measurements of the cAMP concentration beneath the netics in cells within intact islets of Langerhans is unknown. plasma membrane ([cAMP]pm) in individual INS-1 b-cells showed that glucagon-like peptide-1(7-36)-amide (GLP-1) RESEARCH DESIGN AND METHODS—The subplasma- induces [cAMP]pm elevation, often manifested as oscillations membrane cAMP concentration ([cAMP]pm) was recorded in a- and b-cells in the mantle of intact mouse pancreatic islets using (9). Glucose has been regarded to only modestly raise islet total internal reflection microscopy and a fluorescent translocation cAMP, supposedly by amplifying the effect of glucagon (10), biosensor. Cell identification was based on the opposite effects but single-cell cAMP recordings have recently shown that of adrenaline on cAMP in a-andb-cells. glucose alone induces marked elevation of cAMP in MIN6 b b RESULTS—In islets exposed to 3 mmol/L glucose, [cAMP] was -cells (11,12) and primary mouse -cells (12,13). Although pm one study reported that the glucose-induced cAMP response low and stable. Glucagon and glucagon-like peptide-1(7-36)-amide 2+ (GLP-1) induced dose-dependent elevation of [cAMP] , often depends on elevation of [Ca ]i (11), other studies show only pm 2+ with oscillations synchronized among b-cells. Whereas glucagon partial or no Ca -dependence of the cAMP signal (12,13). also induced [cAMP]pm oscillations in most a-cells, ,20% of the Like hormone stimulation, glucose induces oscillations of a 2+ -cells responded to GLP-1. Elevation of the glucose concentra- [cAMP]pm, and coordination of the [cAMP]pm and [Ca ]i tion to 11–30 mmol/L in the absence of hormones induced slow elevations generates pulsatile insulin release (12,14). a b [cAMP]pm oscillations in both - and -cells. These cAMP oscil- There are 10 isoforms of cAMP-generating adenylyl lations were coordinated with those of the cytoplasmic Ca2+ con- 2+ cyclases (ACs) with different regulatory properties, nine of centration ([Ca ]i) in the b-cells but not caused by the changes in 2+ which are transmembrane (tm) proteins stimulated by Gas [Ca ]i. The transmembrane adenylyl cyclase (AC) inhibitor 2959- dideoxyadenosine suppressed the glucose- and hormone-induced and the plant diterpene forskolin. Such tmACs mediate the [cAMP]pm elevations, whereas the preferential inhibitors of solu- cAMP-elevating action of glucagon and incretin hormones ble AC, KH7, and 1,3,5(10)-estratrien-2,3,17-b-triol perturbed cell (15). b-cells express multiple tmACs (16), and the Ca2+- metabolism and lacked effect, respectively. stimulated AC8 has been proposed to be particularly important for integrating hormone- and depolarization- CONCLUSIONS—Oscillatory [cAMP]pm signaling in secreta- gogue-stimulated b-cells is maintained within intact islets and evoked signals (17). Soluble AC (sAC) is the only isoform depends on transmembrane AC activity. The discovery of glu- that lacks transmembrane domains. It is insensitive to cose- and glucagon-induced [cAMP]pm oscillations in a-cells indi- forskolin and G-proteins but stimulated by bicarbonate (18) cates the involvement of cAMP in the regulation of pulsatile and Ca2+ (19). Although sAC was first found in the testis, glucagon secretion. Diabetes 60:1535–1543, 2011 it also seems to be expressed in other tissues and was re- cently proposed to be involved in glucose-induced cAMP production in insulin-secreting cells (20). 2+ Like insulin secretion, exocytosis of glucagon from the yclic AMP and Ca are key messengers in the 2+ a-cells is triggered by an increase of [Ca ]i (21). Glucagon regulation of insulin and glucagon secretion release is stimulated by absence of glucose and is maxi- b a from pancreatic - and -cells, respectively, by mally inhibited when the sugar concentration approaches Cnutrients, hormones, and neural factors. Glucose the threshold for stimulation of insulin secretion (22). stimulation of insulin secretion involves uptake and me- Under hypoglycemic conditions, glucagon secretion is also tabolism of the sugar in the b-cells, closure of ATP-sensitive 2+ + 2+ stimulated by adrenaline, which raises [Ca ]i and [cAMP]pm K channels, and depolarization-induced Ca entry gener- via a -andb-adrenergic mechanisms (23,24). There are ating oscillations of the cytoplasmic Ca2+ concentration 1 2+ fundamentally different ideas about the mechanisms un- ([Ca ]i) that trigger periodic exocytosis of secretory gran- derlying glucose inhibition of glucagon secretion, including ules (1,2). This process is amplified by mechanism(s) fl b d – 2+ paracrine in uences from -and -cells (25 29) and direct acting distal to the elevation of Ca (3). cAMP promotes actions of glucose on the a-cells, resulting in depolarization- exocytosis by facilitating the generation of Ca2+ signals 2+ (30) or hyperpolarization-mediated (22) inhibition of exo- (4,5), by sensitizing the secretory machinery to Ca (4,6), cytosis. Apart from the inhibitory effect of glucose, we observed that very high glucose concentrations unexpectedly stimulate glucagon secretion (31). The stimulatory compo- From the Department of Medical Cell Biology, Biomedical Centre, Uppsala nent may be important under physiological conditions be- University, Uppsala, Sweden. cause the hormone is released in pulses from rat (32) and Corresponding author: Anders Tengholm, [email protected]. Received 3 August 2010 and accepted 21 February 2011. human (33) islets. Glucose thus causes alternating periods of DOI: 10.2337/db10-1087 stimulation and inhibition resulting in time-average reduction Ó 2011 by the American Diabetes Association. Readers may use this article as of glucagon secretion. Ca2+ is probably not the only mes- long as the work is properly cited, the use is educational and not for profit, and the work is not altered. See http://creativecommons.org/licenses/by senger in glucose-regulated glucagon release (29). Like for -nc-nd/3.0/ for details. insulin secretion, cAMP is believed to promote glucagon diabetes.diabetesjournals.org DIABETES, VOL. 60, MAY 2011 1535 cAMP OSCILLATIONS IN PANCREATIC ISLETS release by enhancing intracellular Ca2+ mobilization, Ca2+ 30 min. After rinsing with Tris buffer, the MACH 3 rabbit probe alkaline influx through the plasma membrane, and mobilization of phosphatase polymer kit (Biocare Medical, Concord, CA) was used for visu- ’ secretory granules (23,24,34,35). However, it has also been alization according to the manufacturer s instructions. The reaction was N 2+ stopped by addition of Tris buffer. After rinsing with distilled water, cell nuclei suggested that cAMP-mediated reduction of -type Ca were stained with hematoxylin (Histolab) for 0.5–2 min. currents can explain the inhibitory effect of GLP-1 on Glucose oxidation. Triplicate vials, each containing 104 MIN6 b-cells and glucagon secretion (36). Krebs-Ringer bicarbonate–HEPES buffer (100 mL) supplemented with 3.3 Until now, nothing was known about cAMP kinetics in mmol/L (94 GBq/mol) or 11.1 mmol/L (28 GBq/mol) D-[U-14C]glucose, were a-cells and all information on primary b-cells was based on incubated with or without test compounds for 90 min at 37°C under an at- studies of dispersed islet cells. However, as a result of gap mosphere of 95/5% O2/CO2 during slow shaking. Metabolism was then stopped by addition of 17 mmol/L antimycin A (Sigma-Aldrich) in ethanol. The gener- junctional coupling and paracrine influences, the electro- 14 m 3 2+ ated CO2 was trapped in 250 L hyamine 10 (Perkin-Elmer) during in- physiological characteristics and [Ca ]i signaling in intact cubation at 37°C for 2 h as previously described (38). Radioactivity was islets differ considerably from those in dispersed b-cells measured in a liquid scintillation counter after addition of 5 mL Ultima Gold (2). Therefore, the aim of the current study was to clarify scintillation fluid (Perkin-Elmer). how glucose, glucagon, and GLP-1 affect cAMP signaling in Image and statistical analysis. Image analysis was made using the MetaFluor or ImageJ (W.S. Rasband, National Institutes of Health, http://rsb.info.nih.gov/ a- and b-cells within intact islets of Langerhans. 2+ ij) software. [cAMP]pm and [Ca ]i response magnitudes were calculated from time-average data obtained before and during the stimulation period. Data are RESEARCH DESIGN AND METHODS presented as means 6 SEM. Statistical comparisons were assessed with Stu- Adrenaline, glucagon, GLP-1, 2959-dideoxyadenosine (DDA), EGTA, poly-L- dent t test. lysine, 3-isobutylmethylxanthine (IBMX), somatostatin, and HEPES were from Sigma. Catechol estrogen (CE) [1,3,5(10)-estratrien-2,3,17-b-triol] for AC in- hibition and the inactive control compound 1,3,5(10)-estratrien-3,17-b-triol RESULTS were from Makaira Limited (London, U.K.). KH7 was a gift from Drs. J. Buck Adrenaline has opposite effects on [cAMP]pm in a- and L. Levin, Weill Medical College of Cornell University, New York, NY. The b A 2+ and -cells. Figure 1 illustrates TIRF measurements on acetoxymethyl esters of the Ca indicators Fura-PE3 and Fura Red were peripheral mouse islet cells expressing the cAMP biosensor, obtained from TEFLabs (Austin, TX) and Invitrogen, respectively. Adenovi- B fl ruses expressing a fluorescent cAMP biosensor have previously been de- and Fig. 1 shows TIRF images of the uorescence-labeled scribed (12). subunits of the cAMP indicator. The CFP-to-YFP ratio was Islet isolation, cell culture, and virus infection.