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Islet Studies

TGR5 Activation Promotes Stimulus-Secretion Coupling of Pancreatic β-Cells via a PKA-Dependent Pathway

  1. Jonas Maczewsky1,
  2. Julia Kaiser1,
  3. Anne Gresch2,
  4. Felicia Gerst3,
  5. Martina Düfer2,
  6. Peter Krippeit-Drews1 and
  7. Gisela Drews1⇑
  1. 1Institute of Pharmacy, Department of Pharmacology, Eberhard Karls University of Tübingen, Tübingen, Germany
  2. 2Institute of Pharmaceutical and Medicinal Chemistry, Department of Pharmacology, University of Münster, Münster, Germany
  3. 3Institute for Diabetes Research and Metabolic Diseases, Helmholtz Center Munich, Eberhard Karls University of Tübingen, Tübingen, Germany
  1. Corresponding author: Gisela Drews, gisela.drews{at}uni-tuebingen.de
Diabetes 2019 Feb; 68(2): 324-336. https://doi.org/10.2337/db18-0315

Abstract

The Takeda-G-protein-receptor-5 (TGR5) mediates physiological actions of bile acids. Since it was shown that TGR5 is expressed in pancreatic tissue, a direct TGR5 activation in β-cells is currently postulated and discussed. The current study reveals that oleanolic acid (OLA) affects murine β-cell function by TGR5 activation. Both a Gαs inhibitor and an inhibitor of adenylyl cyclase (AC) prevented stimulating effects of OLA. Accordingly, OLA augmented the intracellular cAMP concentration. OLA and two well-established TGR5 agonists, RG239 and tauroursodeoxycholic acid (TUDCA), acutely promoted stimulus-secretion coupling (SSC). OLA reduced KATP current and elevated current through Ca2+ channels. Accordingly, in mouse and human β-cells, TGR5 ligands increased the cytosolic Ca2+ concentration by stimulating Ca2+ influx. Higher OLA concentrations evoked a dual reaction, probably due to activation of a counterregulating pathway. Protein kinase A (PKA) was identified as a downstream target of TGR5 activation. In contrast, inhibition of phospholipase C and phosphoinositide 3-kinase did not prevent stimulating effects of OLA. Involvement of exchange protein directly activated by cAMP 2 (Epac2) or farnesoid X receptor (FXR2) was ruled out by experiments with knockout mice. The proposed pathway was not influenced by local glucagon-like peptide 1 (GLP-1) secretion from α-cells, shown by experiments with MIN6 cells, and a GLP-1 receptor antagonist. In summary, these data clearly demonstrate that activation of TGR5 in β-cells stimulates insulin secretion via an AC/cAMP/PKA-dependent pathway, which is supposed to interfere with SSC by affecting KATP and Ca2+ currents and thus membrane potential.

Introduction

In recent years, it became evident that the membrane protein Takeda-G-protein-receptor-5 (TGR5), also known as GPBA, MBAR, or Gpbar1, plays an important role in energy and glucose metabolism (1,2). TGR5 is present in several tissues and cell types including heart, spleen, intestine, macrophages, and pancreas (3,4). The receptor is involved in physiological processes such as inflammation, gallbladder filling, gastrointestinal motility, and thermogenesis (1,5–7). TGR5 stimulates secretion of glucagon-like peptide 1 (GLP-1) from intestinal L cells and thus regulates glucose metabolism (8–10). TGR5 activation leads to energy expenditure, which in turn improves glucose homeostasis (9). Noteworthy, after vertical sleeve gastrectomy, TGR5 contributes to the beneficial effects of the surgery (11).

The endogenous ligands of the receptor are bile acids that potently regulate glucose homeostasis (3). For oleanolic acid (OLA), a triterpene isolated from Olea europaea that improves metabolic disorders and has antidiabetes effects (12,13), the situation is less clear. While OLA is proposed to be a TGR5 agonist in pancreatic islets by one study (4), another group excludes an increase in cAMP concentration by OLA normally observed downstream of TGR5 activation (14). In addition, direct effects of OLA on β-cells through increased acetylcholine levels and the muscarinic M3 receptor were reported (15).

Since it was discovered that TGR5 is present in pancreatic β-cells, several in vitro studies described a direct effect of TGR5 on islet cell function (4,16–18). First, Kumar et al. (4) showed the stimulating effect of TGR5 agonists on insulin secretion in β-cells by an increase of intracellular Ca2+ concentration ([Ca2+]c) due to Ca2+ release from intracellular stores. They postulated a pathway through cAMP, exchange protein directly activated by cAMP (Epac), and phospholipase C (PLC) (4). In contrast, another study found that activation of TGR5 by the bile acid tauroursodeoxycholic acid (TUDCA) stimulates insulin secretion via protein kinase A (PKA) (16). This pathway was associated neither with changes in the activity of KATP channels nor with modified Ca2+ signals but included an increase of cAMP, activation of PKA, and phosphorylation of cAMP response element–binding protein (CREBP) (16). Both studies demonstrated TGR5 activation in clonal and murine β-cells, respectively. However, the results point to completely different cAMP-mediated signaling pathways.

It cannot be ruled out that some effects of TGR5 agonists are mediated by the farnesoid X receptor (FXR). Some bile acids are able to rapidly activate a nongenomic FXR-dependent pathway in β-cells (19). FXR and TGR5 are known to influence each other after ligand binding. However, the exact mechanism of this interaction has not been clarified yet (20).

Another in vitro study suggests that stimulating effects of TGR5 agonists in the pancreas are mainly due to GLP-1 released from α-cells that acts in a paracrine manner on β-cells (18). As with GLP-1, activation of TGR5 improves mass and function of β-cells in diabetic mouse models (21). Thus, TGR5 agonists might have a promising therapeutic profile (12).

Taken together, the potential of TGR5 to influence glucose metabolism has been shown in several studies. However, the precise pathways and contribution of different islet cells and peripheral organs are still a matter of debate. Therefore, in the current study the direct effects of OLA and two well-known TGR5 agonists on β-cell stimulus-secretion coupling (SSC) were investigated.

Research Design and Methods

Cell and Islet Preparation

Details are described by Gier et al. (22). In brief, mouse islets were isolated by injecting collagenase (0.5–1 mg/mL) into the pancreas and by handpicking after digestion at 37°C. Male and female wild-type C57Bl/6 (WT) mice were used in equal shares. FXR knockout (FXR−/−) mice and Epac2 knockout (Epac2−/−) mice are all on a C57Bl/6 background and were housed under same conditions. Mice were bred in the animal facility of the Department of Pharmacology at the University of Tübingen. Principles of laboratory animal care (NIH publication no. 85-23, revised 1985) and German laws were followed. Human islets were provided, by JDRF award 31-2008-416 (European Consortium for Islet Transplantation Islet for Basic Research Program), from the Islet Transplantation Centre (Milan, Italy). Mouse and human islets were dispersed to single cells and cell clusters, respectively, by trypsin treatment.

Solutions and Chemicals

Recordings of [Ca2+]c were performed with a bath solution that contained (in mmol/L) 140 NaCl, 5 KCl, 1.2 MgCl2, 2.5 CaCl2, glucose as indicated, and 10 HEPES, pH 7.4, adjusted with NaOH. The same bath solution was used for patch clamp measurements to record KATP current and membrane potential (Vm) in the perforated patch configuration. For Ca2+ current measurements in the perforated patch configuration, bath solution consisted of (in mmol/L) 115 NaCl, 1.2 MgCl2, 10 CaCl2, 10 tetraethylammonium chloride, 10 HEPES, 15 glucose, and 0.1 tolbutamide, pH 7.4, adjusted with NaOH. Krebs-Ringer HEPES solution (KRH) for insulin secretion was composed of (in mmol/L) 120 NaCl, 4.7 KCl, 1.1 MgCl2, 2.5 CaCl2, glucose as indicated, 10 HEPES, and 0.5% BSA, pH 7.4, adjusted with NaOH. Pipette solution for cell-attached KATP current and Vm recordings consisted of (in mmol/L) 10 KCl, 10 NaCl, 70 K2SO4, 4 MgCl2, 2 CaCl2, 10 EGTA, 20 HEPES, and 0.27 amphotericin B, pH, adjusted to 7.15 with KOH. For determination of the Ca2+ currents, pipette solution was composed of (in mmol/L) 10 KCl, 10 NaCl, 7 MgCl2, 70 Cs2SO4, 10 HEPES, and 0.27 amphotericin B, with pH adjusted to 7.15 with NaOH. Murine islet cell clusters and islets were cultured in RPMI 1640 (11.1 mmol/L glucose) enriched with 10% FCS and 1% penicillin/streptomycin. MIN6 cells were incubated in DMEM containing 22.2 mmol/L glucose, 15% FCS, and 1% penicillin/streptomycin. Human islets were kept in Connaught Medical Research Laboratories medium with 5.5 mmol/L glucose.

OLA and NF449 were obtained from Biomol (Hamburg, Germany). Fura-2-acetoxymethyl ester (Flura-2-AM) was purchased from Biotrend (Köln, Germany). Edelfosine and myristoylated protein kinase A inhibitor 14-22 amide (Myr-PKI) were from Tocris (Wiesbaden, Germany). RPMI 1640 medium, FCS, penicillin/streptomycin, and trypsin were from Invitrogen (Karlsruhe, Germany). DMEM medium was from Biozym Scientific (Hessisch Oldendorf, Germany). TUDCA was obtained from Merck (Darmstadt, Germany) and exendin (9-39) amide (exendin 9-39) from Bachem (Bubendorf, Switzerland). The cAMP ELISA kit was from Cayman Chemical, Ann Arbor, MI. All other chemicals were purchased from Sigma-Aldrich (Deisenhofen, Germany) or Merck in the purest form available.

Measurement of [Ca2+]c

Details have previously been published (22). In brief, cells were loaded with 5 μmol/L Fura-2-AM for 35 min at 37°C. Fluorescence was excited at 340 and 380 nm and emission filtered (LP515) and measured by a digital camera. [Ca2+]c was calculated according to an in vitro calibration. The mean [Ca2+]c over 10 min at the end of each interval was calculated to compare [Ca2+]c under different experimental conditions.

Patch Clamp Measurements

Patch pipettes were pulled from borosilicate glass capillaries (Harvard Apparatus, March-Hugstetten, Germany). Ionic currents and Vm were recorded with an EPC-9 patch clamp amplifier using PatchMaster software (HEKA, Lambrecht, Germany). For determination of the KATP current, pulses of 300 ms were performed every 15 s from the holding potential at −70 to −60 and −80 mV, which is the equivalence potential without any current. The amplitude of currents elicited by voltage steps from the holding potential to −60 mV was taken for evaluation. Data of the last three pulses in each interval were averaged and normalized to the control condition. Ca2+ currents were triggered by 100-ms steps from −70 to 0 mV. K+ currents were blocked by tetraethylammonium chloride (TEA), K+-free bath solution, and the KATP channel antagonist tolbutamide. The maximum Ca2+ current was analyzed. For statistics the currents of three succeeding pulses for each measuring point were averaged and data were normalized to the control condition. Vm measurements were evaluated by determination of the plateau potential (from which spikes start) and spike frequency during a 1-min interval after achievement of the maximum OLA effect (minute 4–7 after application). In experiments with KT5720, plateau potential and spike frequency were estimated during 1 min before OLA application and at minute 5–6 after OLA addition.

Insulin Secretion

Details for steady-state incubations have previously been described 23. Briefly, batches of five islets in triplicate were incubated in 1 mL KRH for 1 h at 37°C under conditions indicated. For perifusion experiments, bath chambers were equipped with 50 islets and perifused with KRH under conditions indicated at a rate of 0.7 mL/min at 37°C. Eluate samples were taken every 2 min. MIN6 cells were incubated for 1 h at 37°C under conditions indicated. For determination of the first phase of insulin secretion, AUC was calculated between mins 6 (start of increase) and 21 after the switch to 15 mmol/L glucose.

Insulin was determined by radioimmunoassay (Merck Millipore). Results are presented as the secreted insulin per islet in a specific time. In addition, for MIN6 cells secreted insulin was normalized to the total insulin content and high glucose control condition.

Measurement of cAMP

Batches of 100 islets were incubated in 2 mL KRH for 1 h at 37°C under conditions indicated. Thereafter, buffer was removed and islets were lysed in 0.1 mol/L HCl. Supernatant was used for measuring cAMP by ELISA according to the manufacturer’s protocol.

Statistics

Each series of experiments was performed with islets or islet cells from at least three different mice. Means ± SEM are given for the indicated number of experiments (cell clusters or islets). Statistical significance of differences was assessed by a paired Student t test. Multiple comparisons were made by repeated ANOVA followed by the Student-Newman-Keuls test. P values ≤0.05 were considered significant.

Results

Effect of OLA on [Ca2+]c and Insulin Secretion

The triterpene OLA, extracted from olive leaves, is a powerful modulator of glucose homeostasis (12,13). However, it is not clear by which receptors and downstream pathways OLA interferes with SSC. For evaluation of whether OLA affects β-cell function, its effects on [Ca2+]c and insulin secretion in isolated β-cells and islets, respectively, were investigated. In the presence of 15 mmol/L glucose, [Ca2+]c oscillated. OLA increased mean [Ca2+]c in WT mouse β-cells (Fig. 1A–D). Figure 1E and F reveals that OLA also augmented [Ca2+]c in human β-cells. For evaluation of how this change in [Ca2+]c affects insulin secretion, perifusion experiments with islets of WT mice were performed. Switching from a low to a stimulating glucose concentration evoked the typical biphasic pattern. A first peak secretion (first phase) is followed by consistent release at a lower level (second phase). Addition of 1 μmol/L OLA to the second phase slightly augmented insulin secretion (Fig. 2A). The mean insulin secretion rate for 10 min increased from 22 ± 3 pg insulin/(min × islet) under the control condition to 24 ± 3 pg insulin/(min × islet) (Fig. 2B). In addition to this small, yet significant, effect, the first phase was analyzed in the presence of 1 μmol/L OLA (Fig. 2C). The mean insulin secretion (AUC) during the first 15 min of the first phase of insulin secretion under the high glucose condition was clearly augmented in the presence of 1 μmol/L OLA (37 ± 6 pg insulin/(min × islet)) in comparison with control condition without OLA (27 ± 3 pg insulin/(min × islet)) (Fig. 2D).

Figure 1
Figure 1

OLA increases [Ca2+]c of mouse and human β-cells. A: Representative measurement showing enhancement of glucose-induced oscillations of [Ca2+]c in a mouse β-cell by OLA (1 μmol/L) in the presence of 15 mmol/L glucose. B: Summary of all experiments of this series. C and D: Effect of OLA (10 μmol/L) in the presence of 15 mmol/L glucose. E: Representative experiment showing the effect of 1 μmol/L OLA on a human β-cell in the presence of 10 mmol/L glucose. F: Summary of all experiments of this series. The number in the columns indicates the number of experiments with different cell clusters from three to four mice. Experiments with human β-cells were performed with dispersed islets from one organ donor. *P ≤ 0.05; ***P ≤ 0.001.

Figure 2
Figure 2

OLA stimulates insulin secretion in mouse islets. A: Averaged curve showing the stimulating effect of OLA on insulin secretion in perifusion experiments. OLA (1 μmol/L) was applied during the second phase of insulin secretion. B: Mean insulin secretion rate was analyzed for 10 min before and after addition of OLA in the second phase of insulin secretion. C: For evaluation of the effect on the first phase of insulin secretion, OLA (1 μmol/L) was added before the increase of the glucose concentration. Curves showing the first phase of insulin secretion averaged, with OLA and without OLA. D: Mean insulin secretion rate during the first 15 min of the first phase of insulin secretion in the presence of 15 mmol/L glucose with and without OLA was analyzed. E: Steady-state glucose-induced insulin secretion measured for 1 h is enhanced by 1 and 10 μmol/L OLA but not by 0.1 μmol/L. F: Glucose dependency of the OLA effect (1 μmol/L) on steady-state insulin secretion. The number in the columns indicates the number of experiments with islets from 6–13 mice. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Steady-state insulin secretion comprises both phases of insulin secretion. Application of 1 μmol/L and 10 μmol/L OLA augmented insulin secretion in the presence of 15 mmol/L glucose to 147 ± 11 and 145 ± 16%, respectively (Fig. 2E); 0.1 μmol/L OLA was without effect (113 ± 5%).

The glucose dependency of the drug effect was tested in the presence of 1 μmol/L OLA. OLA did not affect basal insulin secretion at 3 mmol/L glucose but increased it above the threshold concentration for the initiation of insulin secretion (8 mmol/L) and at higher glucose concentrations (Fig. 2F).

Dependence of OLA-Mediated Effects on Gαs and FXR

OLA structurally resembles bile acids. Since acute effects of bile acids in β-cells can be mediated by FXR (19), a possible interaction of the TGR5 agonist with this receptor was investigated. In β-cells of FXR−/− mice, 1 μmol/L OLA increased mean [Ca2+]c similarly to the effect in WT mice (Fig. 3A and B). Accordingly, 1 μmol/L OLA enhanced insulin secretion from islets of FXR−/− mice (Fig. 3C).

Figure 3
Figure 3

A Gαs-coupled receptor but not FXR is the target of OLA. A: Representative trace showing the effect of OLA (1 μmol/L) in the presence of 15 mmol/L glucose on a β-cell of an FXR−/− mouse. B: Summary of all experiments of this series. C: Steady-state glucose-induced insulin secretion from islets of FXR−/− mice is enhanced by OLA (1 μmol/L). D: Inhibition of Gαs by NF449 (10 μmol/L) prevents the stimulating effect of OLA (1 μmol/L) on insulin secretion in islets from WT mice. The number in the columns indicates the number of experiments with different cell clusters or islets from four to six mice. **P ≤ 0.01; ***P ≤ 0.001.

Since the TGR5 is Gs-coupled, the influence of NF449, an inhibitor of the Gαs subunit, on TGR5 activation was investigated to test for this pathway. NF449 (10 μmol/L) did not affect insulin secretion but completely blocked the stimulating effect evoked by OLA in islets of WT mice (Fig. 3D).

Confirmation of the Influence of TGR5 Activation on β-Cell Function by Two Other TGR5 Agonists

The synthetic TGR5 agonist RG239 (1 μmol/L) increased mean [Ca2+]c (Fig. 4A and B) and insulin secretion (Fig. 4C). TUDCA (50 μmol/L), another TGR5 ligand with bile acid structure, provided very similar results for mean [Ca2+]c (Fig. 4D and E) and insulin secretion (Fig. 4F), emphasizing the significance of TGR5 for β-cell function.

Figure 4
Figure 4

Different TGR5 agonists mimic stimulating effects on [Ca2+]c and insulin secretion in β-cells. A: Representative measurement showing enhancement of glucose-induced oscillations of [Ca2+]c by RG239 (1 μmol/L) in the presence of 15 mmol/L glucose. B: Summary of all experiments of this series. C: Steady-state glucose-induced insulin secretion is stimulated by RG239 compared with control islets. D: Representative measurement showing the stimulating effect on glucose-induced oscillations of [Ca2+]c by TUDCA (50 μmol/L) in the presence of 15 mmol/L glucose. E: Summary of all experiments of this series. F: Steady-state glucose-induced insulin secretion is increased by TUDCA. The number in the columns indicates the number of experiments with different cell clusters or islets from three to six mice. *P ≤ 0.05; ***P ≤ 0.001.

Effects of OLA on KATP and Ca2+ Channel Currents

In the well-accepted model of SSC in β-cells, increased [Ca2+]c can result from Ca2+ influx due to closure of KATP channels with subsequent opening of voltage-dependent Ca2+ channels (VDCCs) or to opening of VDCCs. Activity of both channels can be affected by protein kinases (24–26). Patch clamp measurements showed an acute effect on KATP current after OLA administration. In recordings with the perforated patch configuration, 1 μmol/L OLA reduced the KATP current measured in WT β-cells to 54 ± 3% (12.2 ± 1.2 pA) of the control current (100% [22.8 ± 2.4 pA]) in the presence of 0.5 mmol/L glucose (Fig. 5A and B). The effect was dose dependent. The inhibitory effect of 10 μmol/L OLA on the KATP current showed a faster onset of action and reduced the current to 22 ± 2% (7.3 ± 1.3 pA) of the control level (100%, 32.9 ± 5.3 pA) (Fig. 5C and D). The currents were identified as KATP channel currents by use of the KATP opener diazoxide at the end of each measurement.

Figure 5
Figure 5

OLA acutely affects KATP and Ca2+ currents of mouse β-cells. A: Representative experiment showing KATP current measured in the perforated-patch configuration of the patch-clamp technique. Administration of OLA (1 μmol/L) leads to reduction of KATP current in the presence of 0.5 mmol/L glucose. The current is identified as KATP current by the specific KATP channel opener diazoxide (250 μmol/L). B: Summary of all experiments of this series, normalized to the current under control condition. C and D: Increased concentration of OLA (10 μmol/L) amplifies the reduction of the KATP current. E: Currents through VDCCs were measured in the perforated-patch configuration. The representative measurement shows an enhancement of the maximal Ca2+ current during OLA administration compared with control condition in the presence of 15 mmol/L glucose. F: Summary of all experiments of this series at different time points of OLA application, normalized to the current under control condition. The number in the columns indicates the number of experiments with different cell clusters from three to four mice. ***P ≤ 0.001.

As mentioned above, phosphorylation may influence VDCCs. The peak Ca2+ current, measured in the perforated patch configuration, was increased to 118 ± 2% (70.8 ± 9.9 pA) and 122 ± 3% (73.2 ± 10.4 pA) after 2 and 4 min of 1 μmol/L OLA administration, respectively, compared with the control condition (100% [60.5 ± 8.8 pA]) (Fig. 5E and F). At the higher OLA concentration of 10 μmol/L, peak Ca2+ current was augmented after 2 min of drug application but strongly inhibited after 8 min (Fig. 6A and B). OLA (10 μmol/L) also affected second-phase insulin secretion in a biphasic manner (Fig. 6C and D). Evidently, higher concentrations of OLA induce a counterregulating pathway. This observation could explain why 10 μmol/L OLA was not more effective than 1 μmol/L with regard to steady-state insulin secretion (Fig. 2E).

Figure 6
Figure 6

OLA (10 μmol/L) induces a biphasic effect on Ca2+ currents and insulin secretion and depolarized Vm. A: Currents through VDCCs were measured in the perforated patch configuration. The representative measurement shows an enhancement of the maximal Ca2+ current after 2 min of OLA administration (10 μmol/L) compared with the control condition in the presence of 15 mmol/L glucose. Eight minutes of OLA application clearly reduced the current. B: Summary of all experiments of this series at different time points of OLA application, normalized to the current under the control condition. C: Averaged curve showing the transient stimulating effect of 10 μmol/L OLA on the second phase of insulin secretion in perifusion experiments. D: Mean insulin secretion rate was assessed at the time intervals indicated by the symbols. ★, 10 min before OLA application; ▲, 5 min after OLA application; ▼; 10 min before washout OLA-evoked changes in Vm were suppressed by PKA inhibition. E: Representative experiment showing that OLA (10 μmol/L) depolarized Vm and increased action potential frequency. F: Summary of the results concerning the plateau potential. G: Summary of the results concerning spike frequency. AP, action potential. H: Representative experiment in the presence of KT5720 (5 μmol/L) showing that the inhibitor suppresses the OLA effect. I and J: Summary of the results of this series. The number in the columns indicates the number of experiments with different cell clusters or islets from four to six mice. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

At first glance, it may seem astonishing that a clear dual effect of 10 μmol/L OLA on [Ca2+]c is missing (Fig. 1C). However, despite the strong inhibition of Ca2+ currents by 10 μmol/L OLA (Fig. 6), substantial Ca2+ influx may remain. First, KATP current is also markedly reduced by 10 μmol/L OLA (Fig. 5), prolonging the burst time during which Ca2+ channels open (transition from oscillations to a plateau in the presence of 10 μmol/L OLA shown in Fig. 1C). Second, inhibition of KATP current will further depolarize the cells leading to increased opening of Ca2+- and voltage-dependent K+ channels of large conductance (BK channels), resulting in enhanced Ca2+ influx during a single action potential. This assumption is based on the observation that inhibition of BK channels decreases Ca2+ influx (27). Nevertheless, the transient effect of 10 μmol/L OLA on [Ca2+]c can be detected by evaluating maximum Ca2+ concentration. In the experiments presented in Fig. 1, maximum Ca2+ increased from 831 ± 45 nmol/L (n = 26) in the presence of 15 mmol/L glucose to 1,167 ± 75 nmol/L (n = 26, P ≤ 0.001) after application of 10 μmol/L OLA if the usual evaluation procedure (last 10 min of the application interval) was used. However, it amounted to 874 ± 68 nmol/L if only the last 2 min of OLA application was evaluated (n = 26, not significant vs. 15 mmol/L glucose). This shows a clear reduction of maximum Ca2+ concentration over time during OLA application.

KATP and L-type Ca2+ channel currents are two key determinants of the membrane potential of β-cells. Thus, the observed effects on the ion channels should result in changes of the Vm. Figure 6E–G reveals that OLA depolarized Vm and increased the number of action potentials. The described effects were completely suppressed in the presence of the established PKA inhibitor, KT5720 (Fig. 6H–J), pointing to an involvement of this kinase in the OLA-evoked changes in channel activities.

Influence of OLA-Evoked TGR5 Activation on Adenylyl Cyclase and Epac2

The TGR5 belongs to the group of Gs-coupled receptors. The Gαs subunit is known to activate adenylyl cyclase (AC), which leads to cAMP production (3). For verification of this pathway for OLA, the AC inhibitor 2′5′-dideoxyadenosine (DDA) was used in insulin secretion experiments. Remarkably, DDA (100 μmol/L) alone had a stimulating effect on insulin secretion (Fig. 7A). In the presence of DDA, OLA no longer stimulated insulin secretion but, rather, reduced it. Furthermore, OLA (1 μmol/L) increased the intracellular cAMP concentration by ∼23% in five of six experiments (Fig. 7B). In one experiment, we observed a paradoxical decrease of ∼10%.

Figure 7
Figure 7

Inhibition of the AC but not the knockout of Epac2 prevents the effect of OLA. A: Inhibition of the AC with DDA (100 μmol/L) prevents the stimulatory effect of OLA (1 μmol/L) on insulin secretion of islets from WT mice. B: OLA (1 μmol/L) increases the intracellular cAMP concentration in islets from WT mice in the presence of 15 mmol/L glucose. C: Effect of OLA (1 μmol/L) on [Ca2+]c in the presence of 15 mmol/L glucose in β-cells from Epac2−/− mice. D: In islets of Epac2−/− mice, steady-state glucose-induced insulin secretion is increased by OLA (1 μmol/L) and RG239 (1 μmol/L) compared with control islets. The number in the columns indicates the number of experiments with different cell clusters or islets from three to six mice. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Since Epac is one of the postulated targets of cAMP, islets and β-cells of Epac2−/− mice were used to investigate an involvement of this protein in the OLA-activated pathway. Epac2 is the most abundant isoform in β-cells (28). [Ca2+]c measurements did not reveal any influence of Epac2 on the stimulating effect of 1 μmol/L OLA (Fig. 7C). Moreover, the stimulating effects of OLA and RG239 on insulin secretion were still present in islets from Epac2−/− mice (Fig. 7D).

Involvement of PKA in the Signaling Cascade Downstream TGR5 Activation

For further evaluation of a possible participation of PKA in the TGR5 signaling pathway, the established PKA inhibitors Myr-PKI and KT5720 were tested on OLA-evoked insulin secretion. Myr-PKI itself increased insulin secretion, while KT5720 alone was without a significant effect compared with the respective control conditions in WT islets. Both inhibitors suppressed the stimulatory effect of 1 μmol/L OLA in the presence of 15 mmol/L glucose (Fig. 8A and B). OLA even inhibited insulin secretion under these conditions. Since PLC is supposed to be involved in the TGR5 pathway according to Kumar et al. (4), the PLC inhibitor edelfosine was applied. In contrast to PKA inhibitors, edelfosine (10 μmol/L) did not prevent the stimulation evoked by 1 μmol/L OLA (Fig. 8C [same controls as in Fig. 8A]).

Figure 8
Figure 8

Stimulating effects of OLA on insulin secretion are mediated by PKA but not by PLC or PI3K. Experiments were performed with islets from WT mice. A: The PKA antagonist Myr-PKI (1 μmol/L) eliminates the increasing effect of OLA. B: Another PKA antagonist, KT5720 (5 μmol/L), also prevents the stimulation by OLA. C: The PLC antagonist edelfosine (10 μmol/L) does not influence the effect of OLA. D: The PI3K inhibitor wortmannin (100 nmol/L) does not reduce the stimulation by OLA. The number in the columns indicates the number of experiments with different islets from six mice. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Phosphoinositide 3-kinase (PI3K) is also proposed to be involved in the signaling pathway downstream of TGR5 activation. The PI3K inhibitor wortmannin did not prevent but even amplified the OLA effect on insulin secretion (Fig. 8D [same controls as in Fig. 8B]). Neither edelfosine nor wortmannin alone changed insulin secretion induced by 15 mmol/L glucose.

α-Cells and GLP-1 Are Not Involved in Effects of OLA in β-Cells

α-Cells can secrete GLP-1, and activation of TGR5 is known to increase GLP-1 production and secretion (18). For exclusion of the possibility that GLP-1 contributes to the OLA-evoked effects in β-cells, the GLP-1 receptor antagonist exendin 9-39 was used in secretion experiments. Exendin 9-39 did not prevent the stimulating effect of 1 μmol/L OLA on insulin secretion (Fig. 9A). The potency of the inhibitor exendin 9-39 at the GLP-1 receptor was proved by the fact that the stimulation of 50 nmol/L GLP-1 was completely blocked by the antagonist (Fig. 9B [same controls as in Fig. 9A]). Notably, 100 nmol/L exendin 9-39 alone did not significantly alter the amount of secreted insulin. For circumvention of a possible influence of α-cells, the β-cell line MIN6 was used to perform insulin secretion experiments. Insulin secretion of MIN6 cells was dependent on the glucose concentration (insulin levels reached 26.6 ± 2.9% at a low glucose concentration compared with levels at a stimulatory concentration of 15 mmol/L glucose). Application of 1 and 10 μmol/L OLA significantly increased insulin secretion to 111.5 ± 3.0 and 121.4 ± 7.8%, respectively, compared with 15 mmol/L glucose alone (Fig. 9C). These experiments support the assumption that the TGR5 agonist OLA acts directly on β-cells.

Figure 9
Figure 9

The stimulating effect of OLA on β-cells is not mediated by GLP-1 from α-cells. A: Inhibition of the GLP-1 receptor by the antagonist exendin 9-39 (100 nmol/L) in WT islets does not influence the OLA-mediated increase of insulin secretion. B: The potential of exendin 9-39 (100 nmol/L) to inhibit the GLP-1 receptor is demonstrated by the abolishment of the GLP-1–induced (50 nmol/L) increase in insulin secretion. C: In MIN6 cells, glucose-induced insulin secretion (15 mmol/L glucose) is increased by OLA. The number in the columns indicates the number of experiments. Islets for the series with exendin 9-39 were from seven different mice. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Discussion

OLA Directly Stimulates β-Cells by Binding to the TGR5

During the last decades, it became evident that TGR5 agonists are important contributors to the regulation of glucose metabolism. Several studies have shown reduction of blood glucose concentration and improvement of the energy expenditure by TGR5 agonists (9,12,29). TGR5 activation in L cells crucially affects GLP-1 secretion (30). Here, we identify OLA as a TGR5 agonist of β-cells and show a stimulating effect of OLA and two other TGR5 agonists, RG239 and TUDCA, on islets of Langerhans in vitro excluding factors like GLP-1 secreted from L cells. TGR5 activation concurrently affects several parameters of SSC including current through KATP channels and VDCCs and, as a result, Vm. Changes in the activity of these channels are followed by enhanced [Ca2+]c and insulin secretion. Kumar et al. (4) also demonstrated a direct effect on isolated β-cells after TGR5 agonist administration. In their study, OLA leads to enhanced glucose-induced insulin secretion; however, an increase in [Ca2+]c is only shown at substimulatory glucose concentration and is attributed to release from intracellular Ca2+ stores. This effect cannot account for increased insulin secretion after stimulation of β-cells with glucose. In contrast, our data clearly show that enhanced Ca2+ influx contributes to increased [Ca2+]c after TGR5 activation at a stimulatory glucose concentration and not at basal one.

In α-cells, alternative splicing of proglucagon enables synthesis and secretion of GLP-1 (17). Kumar et al. (18) observed an increased GLP-1 secretion from pancreatic α-cells after TGR5 activation. They suggest that this is mediated via the cAMP/Epac/PLC-dependent pathway. Moreover, synthesis of GLP-1 in α-cells was stimulated by the cAMP/PKA/phosphorylated CREBP cascade. The authors provide evidence that this pathway is activated by hyperglycemia (18). To examine a possible GLP-1–mediated stimulation of insulin secretion after TGR5 activation under physiological conditions, we blocked the GLP-1 receptor by the antagonist exendin 9-39 (31). Since exendin 9-39 did not prevent the effect of OLA, we conclude that OLA stimulates insulin secretion independent of GLP-1 receptor activation. Kumar et al. (18) performed a similar experiment with human islets but with a higher concentration of exendin 9-39 and after culturing the islets in the presence of 25 mmol/L glucose for 7 days. They claim that in this glucotoxic model, exendin 9-39 reduces the effect of the TGR5 agonist INT-777; however, this reduction is marginal and insulin release is still approximately twofold higher compared with the effect of glucose alone. Our view that the effects of TGR5 agonists are not mediated by GLP-1 released by α-cells is further supported by the observation that OLA increased insulin secretion of MIN6 cells. The MIN6 cell line solely consists of clonal β-cells, and an effect of locally secreted GLP-1 from other cell types can be ruled out (32).

TGR5 agonists are structural analogs of bile acids, the endogenous activators of TGR5. Bile acids acutely affect additional targets regulating glucose metabolism, particularly the FXR (19,33). In β-cells, Vettorazzi et al. (16) found that TUDCA activates a TGR5-dependent pathway and suggested that FXR is not involved. In the current study, the involvement of FXR was excluded owing to experiments with a FXR−/− mouse model. Teodoro et al. (14) also showed a stimulating effect of OLA on insulin secretion but excluded increased cAMP concentration and thus involvement of the TGR5 as explanation for this observation. In contrast, our results with inhibitors of the Gαs subunit and the AC clearly indicate a TGR5-dependent pathway for OLA.

OLA Acts via a cAMP/PKA-Dependent Pathway

The TGR5/Gαs/AC pathway results in increased cAMP concentrations (5,34). This fits well with the OLA-induced increase of the cAMP concentration and the loss of efficacy of OLA after inhibition of AC in our experiments. Enhanced cAMP levels are known to activate PKA and/or Epac, which is also described for β-cells (4,16,30). Kumar et al. (4) exclude any influence of OLA on PKA in β-cells but describe a pathway via Epac, followed by PLC activation, which leads to enhanced insulin secretion. This suggestion is based on a single series of secretion experiments with MIN6 cells and the high concentration of 50 μmol/L OLA (4). Moreover, Kumar et al. (4) blocked the effect of 50 μmol/L OLA with the PLC antagonist U73122. However, the used concentration of U73122 can exert unspecific effects, such as modulation of transient receptor potential melastatin 3/4 (TRPM3/4) channels as well as stimulation of inositol triphosphate synthesis and mobilization of Ca2+ from intracellular stores (35–37).

To clarify the discrepancy in our results, we used an Epac2−/− mouse model. Epac2 is more abundant compared with Epac1 and is an important target of cAMP in β-cells (28,30). Since TGR5 agonists effectively enhanced [Ca2+]c and insulin secretion in β-cells and islets of Epac2−/− mice, an involvement of Epac2 seems to be unlikely. Nevertheless, a possible influence of Epac1 should be considered. Likewise, PLC blockade by edelfosine did not suppress the OLA effect on insulin secretion, also speaking against an involvement of the Epac/PLC pathway after TGR5 activation.

PI3K has been identified as another downstream target of Epac in processes like angiogenesis or stem cell differentiation (38,39). The PI3K inhibitor wortmannin revealed that PI3K seems not to be involved in stimulating effects of OLA.

We identified PKA as the downstream kinase in the TGR5/cAMP pathway. OLA completely lost the stimulating effect on insulin secretion after PKA inhibition with two different PKA inhibitors, Myr-PKI and KT5720. This is supported by findings of Vettorazzi et al. (16), who showed that the TGR5 agonist TUDCA was ineffective in stimulating insulin secretion in the presence of the PKA inhibitor H89. Worth mentioning, the link between cAMP and PKA is clearly demonstrated for the GLP-1 pathway in β-cells (40,41).

The puzzling observation that inhibition of AC or PKA leads to stimulation of insulin secretion may be due to a cross talk between cAMP and cGMP as described for other organs (42,43), especially activation of a cGMP-specific PDE by PKA (44,45). Inhibition of PKA would thus increase cGMP concentration, leading to protein kinase G (PKG)-dependent closure of KATP channels (46). Inhibition of the AC by DDA would also reduce PKA activity with similar consequences at least for the cGMP/PKG/KATP channel signaling pathway. Remarkably, during inhibition of the AC/cAMP/PKA pathway, OLA is not without effect but exerts inhibition of insulin secretion. This is most likely due to the biphasic effect of OLA. Apparently, after inhibition of the stimulatory pathway the inhibitory one that is PKA independent prevails.

KATP and VDCCs Mediate Stimulating Effects of OLA

Although presenting results in favor of the PKA pathway, Vettorazzi et al. (16) did not find any changes in KATP channel activity or [Ca2+]c after TGR5 activation in β-cells. In our experiments, OLA caused both a distinct reduction of the KATP current and an increase in Ca2+ current, probably due to phosphorylation of both channel proteins by PKA. After PKA activation, KATP channel activity is reduced, resulting in membrane depolarization and enhanced insulin secretion (24). Suitably, OLA-evoked changes in Vm, which are a result of the effects on the channels, are suppressed by the PKA inhibitor KT5720.

The VDCC in pancreatic islet cells is a possible target to control insulin secretion (47). Phosphorylation of VDCCs could affect channel activity, thus increasing the Ca2+ current (25,26). It is worth mentioning that the effect of OLA on VDCCs is not secondary to inhibition of KATP channels, since the latter were not functional under the relevant experimental conditions. Thus, KATP channel closure and VDCC activation together cause increased [Ca2+]c followed by enhanced insulin secretion. Since OLA increases [Ca2+]c in human β-cells, it is to be assumed that the suggested mechanism is also relevant for human β-cells. The proposed direct interaction of PKA with ion channels would result in a rapid effect after TGR5 activation. However, we cannot exclude that other mechanisms besides changes in ion channel activity contribute to the stimulatory effects of TGR5 activation. The cAMP-PKA pathway can directly increase granule exocytosis by enhancing the sensitivity of the exocytotic machinery to Ca2+ (48,49). Two other groups postulated modified protein synthesis by PKA-mediated phosphorylation of CREBP (16,18). Such a mechanism is inconsistent with the rapid effects on SSC starting within seconds. However, protein synthesis may account for effects on exocytosis after prolonged exposure to TGR5 agonists (14,16,18).

In summary, we clearly demonstrated that OLA affects β-cell SSC via TGR5 activation and that TGR5 agonists directly stimulate β-cells to secrete insulin. The data suggest a pathway including AC activation, PKA, closure of KATP, and opening of Ca2+ channels and increased Ca2+ influx. This insulinotropic effect opens new possibilities for pharmaceutical applications of drugs like OLA.

Article Information

Acknowledgments. The authors thank Isolde Breuning for excellent and skillful technical assistance. The authors thank Jelena Sikimic for performing some of the experiments with human islet cells and Friederike Anna Steudel and Nia Blackwell for careful and excellent revision of the manuscript, all from Institute of Pharmacy, Department of Pharmacology, Eberhard Karls University of Tübingen.

Funding. This work was supported by a grant from the DFG (Deutsche Forschungsgemeinschaft) (DR 225/11-1 to G.D.).

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. J.M. researched data and wrote and edited the manuscript. J.K., A.G., and F.G. researched data. M.D. and P.K.-D. contributed to discussion and study design and edited the manuscript. G.D. designed the study, wrote and edited the manuscript, and contributed to discussion. G.D. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

  • Received March 15, 2018.
  • Accepted October 31, 2018.
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TGR5 Activation Promotes Stimulus-Secretion Coupling of Pancreatic β-Cells via a PKA-Dependent Pathway
Jonas Maczewsky, Julia Kaiser, Anne Gresch, Felicia Gerst, Martina Düfer, Peter Krippeit-Drews, Gisela Drews
Diabetes Feb 2019, 68 (2) 324-336; DOI: 10.2337/db18-0315
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