Redox Signaling is Essential for Insulin Secretion

In this review, we place redox signaling in pancreatic β -cells to the context with signaling pathways leading to insulin secretion, acting for example upon the action of incretins (GLP-1, GIP) and the metabotropic receptor GPR40. Besides a brief description of ion channel participation in depolarization/repolarization of the plasma membrane, we emphasize a prominent role of the elevated glucose level in pancreatic β -cells during glucose-stimulated insulin secretion (GSIS). We focus on our recent findings, which revealed that for GSIS, not only elevated ATP synthesis is required, but also fundamental redox signaling originating from the NADPH oxidase 4- (NOX4-) mediated H 2 O 2 production. We hypothesized that the closing of the ATP-sensitive K + channel (K ATP ) is only possible when both ATP plus H 2 O 2 are elevated in INS-1E cells. K ATP alone or with synergic channels provides an element of logical sum, integrating both metabolic plus redox homeostasis. This is also valid for other secretagogues, such as branched chain ketoacids (BCKAs); and partly for fatty acids (FAs). Branched chain aminoacids, leucine, valine and isoleucine, after being converted to BCKAs are metabolized by a series of reactions resembling β -oxidation of FAs. This increases superoxide formation in mitochondria, including its portion elevated due to the function of electron transfer flavoprotein ubiquinone oxidoreductase (ETF:QOR). After superoxide conversion to H 2 O 2 the oxidation of BCKAs provides the mitochondrial redox signaling extending up to the plasma membrane to induce its depolarization together with the elevated ATP. In contrast, experimental FA-stimulated insulin secretion in the presence of non-stimulating glucose concentrations is predominantly mediated by GPR40, for which intramitochondrial redox signaling activates phospholipase iPLA2 γ , cleaving free FAs from mitochondrial membranes, which diffuse to the plasma membrane and largely amplify the GPR40 response. These events are concomitant to the insulin release due to the metabolic component. Hypothetically, redox signaling may proceed by simple H 2 O 2 diffusion or via an SH-relay enabled by peroxiredoxins to target proteins. However, these aspects have yet to be elucidated.

glucose intake contributes to the increasing pentose phosphate pathway (PPP) supply of NADPH for NADPH oxidase 4 (NOX4), which directly produces H 2 O 2 . The burst of H 2 O 2 then represents a redox signal, which fundamentally determines GSIS, while inducing a cooperative induction of plasma membrane depolarization together with ATP elevation (Figure 1) [1]. The latter originates from the increased ATP synthesis by oxidative phosphorylation (OXPHOS). Hypothetically, either a closure of the ATP-sensitive K + channel (K ATP ) is dependent on both H 2 O 2 plus ATP; or H 2 O 2 activates a synergic channel such as transient receptor potential of the redox shuttles upon GSIS [48]. One may expect that a portion of cytosolic NADPH as a substrate for NOX4 is provided by the glucose-6-phosphate dehydrogenase and also by 6-phosphogluconate dehydrogenase downstream within the PPP, whereas the second portion is generated due to the operation of redox shuttles. These shuttles become more active at higher glucose concentrations and increasingly produce NADPH. NADPH is particularly produced by isocitrate dehydrogenase 1 (IDH1) and malic enzyme 1 (ME1) in the cytosol upon operation of these redox shuttles [48].
In summary, we describe the revisited mechanism of the 1st phase of GSIS as follows. Elevated glucose metabolism and glycolysis allows an increased branching of the metabolic flux, particularly of glucose-6-phosphate G6P, toward PPP, which acts as a predominant source of NADPH. The essential role of PPP was emphasized elsewhere [49]. Amplification of the cytosolic NADPH is also provided by IDH1 and ME1 due to the elevated operation of the three redox shuttles. Since NOX4 was determined as the only NADPH oxidase producing H 2 O 2 directly [50,51], its reaction results in an increase of H 2 O 2 release into the cell cytosol [1]. Finally, the elevated H 2 O 2 , together with concomitantly elevated ATP from the enhanced OXPHOS, is the only way for plasma membrane depolarization up to −50 mV [1]. This threshold subsequently induces Ca L opening, followed by the Ca 2+ influx into the cell cytosol, which in turn induces the exocytosis of insulin granule vesicles. The action potential spikes are then determined by the cycles of Ca L opening, followed by the opening of voltage-dependent channels (K V ) in rodents [52] or calciumdependent (K Ca ) K + -channels in humans. Their action deactivates Ca L, which are, however, again activated in the next Ca L -K V cycle.
Pancreatic β-cells were undoubtedly adapted by phylogenesis to serve as a perfect glucose sensor. The glucose sensing is allowed by several key specific features. At first, specific isoforms of glucose transporters, GLUT2 in rodents and GLUT1 in humans, equilibrate the plasma glucose concentration with the glucose concentration in the cytosol of β-cells [53,54]. Second, a specific isoform IV of hexokinase (also termed glucokinase) cannot be feed-back inhibited by its product glucose-6-phosphate. As a result, there is an efficient unidirectional flux towards the glycolysis [55,56] and, most probably, this allows also branching into the PPP [49]. Originally, the PPP was accounted to utilize only 10% of glucose, due to presumably feed-back inhibition by glucose [57,58]. However, metabolomics studies associated PPP intermediates with GSIS [59], confirming previous studies with various PPP inhibitors [59][60][61][62]. These results collectively demonstrated the important PPP contribution to GSIS. This contribution is also reflected by existing patients having a deficiency of glucose-6-phosphate dehydrogenase associated with the impaired 1st phase of GSIS [63].
The third aspect leading to the perfect glucose sensing lays in the virtual absence of lactate dehydrogenase in β-cells and inefficiency in pyruvate dehydrogenase kinases (PDK) [64]. PDKs would otherwise block pyruvate dehydrogenase (PDH). Thus the highly active PDH and other dehydrogenases, activated also by Ca 2+ influx into the mitochondrial matrix [65], altogether enable that 100% of pyruvate and its equivalents (after pyruvate conversion by transaminases) is utilized by OXPHOS. A minor pyruvate flux ensures anaplerosis of oxaloacetate due to the reaction of pyruvate carboxylase [66]. Its reaction is also important also for the pyruvate/ malate redox shuttle.
The fourth aspect reflects the in vivo inhibitory role of the mitochondrial ATPase inhibitory factor, IF1. IF1 adjusts a proper glucose concentration range for GSIS in rat pancreatic β-cells, INS-1E [67,68]. This is suggested by the demonstration that IF1 silencing allows insulin secretion even at very low glucose approaching to zero in INS-1E cells [67]. In contrast, the IF1 overexpression inhibited GSIS in INS-1E cells [68]. This IF1 role awaits confirmation in vivo.

Plasma membrane events following K ATP closure
Surprisingly, the plasma membrane of β-cells contains up to 60 channels of 16 ion channel families [69]. Moreover, ion channels are also located on the membrane of IGVs to facilitate fusion with the plasma membrane and insulin exocytosis. Resting plasma membrane potential (Vp) is created predominantly by the activity of K + -channels due to a higher concentration of K + inside the β-cell (~150 mM), exceeding that one established outside in capillaries or interstitial fluid (~5 mM). Experimentally, Vp values are measured to be approximately of -75 mV [70]. The K ATP closure then induces depolarization [69,[71][72][73] and activation of Ca L [74]. The action potential firing is the entity that activates Ca L -K V cycles (in rodents), however this firing is initiated by more channel types.
Surprisingly, the action potential firing is not induced until >90% of K ATP channels are closed [75,76]. As a result, only the closure of the remaining ~10% of the K ATP population leads to depolarization [76]. In fact, the activity of the whole K ATP population decreases exponentially with the increasing glucose concentration. Interestingly, 50% of the K ATP population is already closed at 2-3 mM glucose, while Vp remains steady. However, at about 7 mM glucose, 100% of the K ATP ensemble is closed. This is being reflected by the completely vanished K ATP current, which leads to action potential firing [69,70]. This event is termed as a supra-threshold depolarization.
Thus, hyperpolarized interburst phases are induced, while a nearly permanent firing exists at high >25 mM glucose [70]. An intermediate depolarization at 10 mM glucose was reported for mouse β-cells, reversed upon withdrawal of Ca 2+ and Na + , supporting the participation of other channels, such as nonspecific cation channels, contributing to the depolarization (inward) flux [45]. Even an efflux of Cl − was suggested to fulfil this role [77], including the opening of LRRC8/VRAC anion channels [78,79]. The participation of TRPM4 and TRPM5 [80] providing inward currents of certain levels seem to be required for induction of sufficient membrane depolarization together with K ATP closing [46]. This is because the measured resting Vp of -75 to −70 mV is already depolarized by a some extent from the equilibrium Vp equi of −82 mV (5 mM vs. 130 mM [K + ]). The shift is probably due to the opening of nonspecific cation channels, since any of Na + , Ca 2+ and K + can penetrate them. The 100% K ATP closing at higher glucose causes only an insufficient depolarization. Without nonspecific cation channels (or Cl − channels), the established Vp would only be equal to Vp equi , so any shift to −50 mV required for Ca L would not take place. Contribution by the basal opening of other synergic channels is therefore essential. Open synergic channels always induce the inward shift in Vp, so to that depolarization given by 100% K ATP closing reaches −50 mV. This allows opening of Ca L and action potential firing. In summary, besides the heat-activated TRPV1 channel (capsaicin receptor), and TRPV2 or TRPV4, the H 2 O 2 -activated TRPM2 [2], or Ca 2+ -activated TRPM4 and TRPM5 channels belong to the important group of possible synergic channels expressed in β-cells [46].
The same reasoning concerns with anion channels, particularly Cl − channels. The active Cl − transport is provided in β-cells by SLC12A, SLC4A, and SlC26A channels. These channels set the cytosolic Cl − concentration above thermodynamic equilibrium. Besides GABA A , GABA B and glycine receptor Cl − channels considered to be part of the insulin secretion machinery, also volume-regulated anion channels (VRAC) were shown to be open at high glucose. VRACs are heteromers of the leucine-rich repeat containing 8 isoform A (LRRC8A) with other LLRC8 isoforms, forming anion channels [79]. Ablation of LRRC8 in mice led to delayed Ca 2+ responses of β-cells to glucose and diminished GSIS in mice, demonstrating the modulatory role of LRRC8A/VRAC on membrane depolarization leading to Ca L responses [78,79].
Redox Signaling is Essential for Insulin Secretion DOI: http://dx.doi.org /10.5772/intechopen.94312 Upon the action potential firing thus metabolically driven Vp oscillations occur due to the initial glucose rising [69,70]. Cytosolic Ca 2+ oscillations are superimposed from fast (2-60 s periods) and slow (up to several min) Ca 2+ oscillations [81], stemming from Vp oscillations and an interplay with Ca 2+ efflux from the endoplasmic reticulum (ER) [82]. Collectively they lead to pulsatile insulin secretion. The ER involvement is given by the phospholipase C (PLC), responding to the glucosestimulated Ca 2+ influx. PLC produces inositol triphosphate (IP3), which opens the Ca 2+ channel of IP3 receptor (IP3R) of ER; plus diacylglycerol (DAG). Importantly, DAG permits the opening of TRPM4 and TRPM5 via the protein kinase C (PKC) pathway. Another ER Ca 2+ channel, the ryanodine receptor (RyR) may also participate, being activated by ATP, fructose, long-chain acyl-CoAs and cyclic adenosine 5′-diphosphate ribose [81]. Also, the role of other channels was demonstrated for permitting store-operated Ca 2+ entry from ER, particularly of the ternary complex of TRPC1/Orai1/STIM1 [46,83]. TRPC1 belongs to the transient receptor potential canonical (TRPC) family with a modest Ca 2+ selectivity. TRPC1 interacts with Orai1 [84], and in such a functional complex, its channels are activated by STIM1, affecting the amplitude of Ca 2+ oscillations, and correlating with GSIS.
As mentioned above, deactivation of Ca L is ensured by the opening of voltagedependent channels (K V ) in rodents [52] or calcium-dependent (K Ca ) K + -channels in humans. Among the former, tetrameric K V 2.1 is the prevalent form in rodent β-cells. A delayed rectifier K + -current is induced at positive Vp down to −30 mV [85]. The opening of K V 2.1 channels repolarizes Vp and thus closes Ca L channels. Ablation of K V 2.1 thus reduces Kv currents by ~80% and prolongs the duration of the action potential, so more insulin is secreted. Mice with ablated K V 2.1 possess lower fasting glycemia but elevated insulin and reportedly improved GSIS [86]. In contrast, human β-cells use K Ca 1.1 channels (i.e. BK channels) for repolarization of Vp [70]. Note also that downregulation of K V was observed after islet incubation with high glucose for 24 hr [87].

Possible redox regulations of K ATP and other channels
The structure of K ATP has been resolved and numerous mutagenesis studies of K ATP have been conducted. Amino acid residues that are candidate redox targets are yet to be identified. The K ATP channel is a hetero-octamer consisting of four external regulatory sulfonylurea receptor 1 (SUR1, a product of Abcc8 gene) subunits and four pore-forming subunits of potassium inward rectifier, Kir6.2 (Kcnj11 gene) [88,89]. These Kir6.2 subunits cluster in the middle of ~18 nm size structure with a ~13 nm height [90]. The part exposed to the cytosol contains an ATP binding site, located about 2 nm below the membrane. A single ATP molecule was reported to close the channel, i.e. with the other three binding sites left unoccupied [91]. However, the ATP binding site overlaps with the binding site for phosphatidylinositol 4,5-bisphosphate (PIP 2 ), which stabilizes the open state. Palmitoylation of Cys166 of Kir6.2 was then reported to amplify the responsiveness to PIP 2 [92]. Upon the release of PIP 2 from the binding site, the open probability becomes decreased [90,93,94].
Diazoxide or cromakalim, as well as numerous other openers, set K ATP pharmacologically in the open state even at a high ATP concentration [95]. In contrast, the artificial K ATP closing by sulfonylurea derivatives, such as glibenclamide, takes place independently of ATP. Besides this sulfonylurea binding site, each of the four SUR1 subunits contains MgATP and MgADP binding sites. MgATP is hydrolyzed at the nucleotide binding fold 1 (NBF1) to MgADP. Resulting MgADP subsequently activates K ATP at NBF2. This is indeed reflected by the ATP-sensitive increase in K + conductance and following lower excitability, accompanied by the lower sensitivity to ATP inhibition [91].
The phosphorylation of K ATP reportedly sets the sensitivity of the K ATP ensemble. The setting is such that transitions upon the glucose rise from 3 or 5 mM to 7 mM or > 10 mM result in the closing of the remaining 10% of the initially open channels by elevations between just the two ATP concentrations falling into the mM range. Any redox component in this was never indicated and should be studied. Nevertheless, phosphorylation mediated by the protein kinase A (PKA) was already reported to act in this unusual setting. Thus Thr224 [96] and Ser372 were reported to be the verified PKA phosphorylation sites. Their phosphorylation increases the open probability of K ATP [97]. This might hypothetically provide closing mechanism acting at higher ATP concentration or even requiring H 2 O 2 . In a longer time scale, phosphorylation also increases the number of channels in the plasma membrane. Also, Thr224 was found to be phosphorylated by Ca 2+ and calmodulin-dependent kinase II (CaMKII) while interacting with β IV -spectrin [98]. In vivo, also autonomic innervations and paracrine stimulation ensure sufficient PKA-mediated phosphorylation of K ATP .
Since the original discovery of the essential role of K ATP in GSIS [99], only an indirect inhibition of K ATP by H 2 O 2 was observed in smooth muscle cells [100]. Nevertheless, other redox-sensitive targets have been identified in pancreatic β-cells. But we can exclude the possibility that the IGV exocytosis itself might be directly induced by H 2 O 2 , independently of Ca 2+ [52], since the ability of exogenous H 2 O 2 to induce insulin secretion in INS-1E cells was only partially blocked by NOX4-siRNA, but it was completely blocked by a Ca L blocker nimodipine [1]. Consequently, albeit the used H 2 O 2 doses exceeded 100 μM, they did not directly stimulate the K ATP -independent exocytosis of insulin granules.
A second possibility would be that Ca L channels themselves may be hypothetically co-activated by H 2 O 2 . Third, the plasma membrane depolarization might be redox sensitive, so that H 2 O 2 could directly or indirectly inhibit repolarizing K + -channels, such as K V [101][102][103]. The fourth plausible redox link with GSIS would concern with the reported redox activation of TPRM2 depolarizing channels [2]. The latter is the most plausible, since it is related to a Ca 2+ -induced [52,104] or H 2 O 2 -induced exocytosis of insulin granules by the H 2 O 2 -activation of TPRM2 depolarizing channels [2,105]. Note, our results excluded the Ca 2+ -independent H 2 O 2 -induced exocytosis of IGVs at least in rat pancreatic β-cells [1]. Therefore, if the H 2 O 2 -activated TRPM2-dependent mechanism exists, it must provide the required synergy with K ATP , to reach the −50 mV plasma membrane depolarization threshold. Note also, that TRPM2 was already implicated as a significant player in the GLP-1 potentiation of insulin secretion [106].
Finally, a competition for NADPH between NOX4 and a hypothetical NADPHactivated K + -channel could exist. Nevertheless, using patch-clamped INS-1E cells in a whole cell mode, we demonstrated a closure of K ATP by H 2 O 2 produced by NOX4 at high glucose, since in cells silenced for NOX4, even ATP resulting from the metabolism of high glucose was unable to close the K ATP channel [1].
Let us emphasize downstream pathways that are important for acute effects in pancreatic β-cells, which predominantly lead to either modulation of the plasma membrane channels, typically Ca L , K ATP and K V , so to ensure more intensive insulin secretion; or their action evokes stimulation of insulin secretion via ensuring the surplus Ca 2+ influx to the cytosol from ER or mitochondria; or, else, their action targets proteins of the exocytotic machinery on the IGV or plasma membranes. The latter responses alter the kinetics of IGVs in docking, priming and fusion with the plasma membrane, so to facilitate exocytosis. Interestingly, these events could be independent of Ca L and theoretically could take place at low glucose concentrations.
Activation of Gαs increases the activity of transmembrane adenylate cyclases (tmAC) producing cAMP from ATP [108,109]. A number of phosphodiesterases (of 11 families) degrade cAMP (some also or exclusively cGMP). cAMP is a universal 2nd messenger having a specific function in amplifying of GSIS and insulin secretion stimulated with other secretagogues. Also, soluble adenylate cyclases (sACs) exist, notably in the mitochondrial matrix, while their reaction is potentiated by Ca 2+ and bicarbonate. The major mediators of cAMP effects are cAMP-dependent PKA [114], including PKA tethered to the outer mitochondrial membrane [115,116], and the parallel pathway of enhanced signaling via exchange proteins directly activated by cAMP 2 (EPAC2) [117][118][119].
In pancreatic β-cells, the PKA pathway is involved in signaling of incretin (GLP-1 and GIP) receptors [107,120]. It exerts a minor contribution to signaling from metabotropic receptors, such as GPR40, which is sensing long chain fatty acids [111]. PKA typically amplifies the Ca 2+ -dependent exocytosis of insulin granules. The core pathway involves PKA phosphorylation and hence activation of the Ca L β2-subunit, in concert with K ATP phosphorylation decreasing the ATP concentration range required for its closure (see above) [121]. In addition, PKA inhibits Kv channels, which otherwise terminate plasma membrane depolarization; hence this prolongs already more intensive Ca 2+ influx via phosphorylated Ca L and hence exocytosis of insulin granules [122].
Another PKA target is the exocytosis-modulating protein termed snapin, the phosphorylation of which allows its interaction with the other IGV proteins, which enhances the 1st GSIS phase [123]. Snapin participates in tethering of IGVs to the plasma membrane by coiled-coil interaction with a lipid-anchored protein SNAP-25 [124].
Altogether, the PKA pathway ensures about 50% of cAMP responses in β-cells [125], while the EPAC2 pathway ensures the remaining responses [117][118][119]. EPAC2 protein possesses a guanine nucleotide exchange activity, thus inducing the Ca 2+induced Ca 2+ release from ER via RyR [126] (questioned in [127]), occurring only at high glucose, since it requires the primary Ca L opening [128], which also partially refills the ER Ca 2+ stores. The EPAC2 pathway also affects the IGV proteins and thus facilitates the insulin exocytosis. For example, Rim2a protein is a target [129,130], located on the inner plasma membrane surface and on IGVs, representing a scaffold for IGV exocytosis [131]. Rim2a interacts with Rab3A of IGVs and the resulting Rim2a-Rab3A complex facilitates docking of IGVs into the plasma membrane. This is followed by so-called priming, which is subsequently initiated by the Rim2a interaction with the Munc13-1 protein. Munc13-1 then opens syntaxin 1 from its closed conformation, thus allowing fusion with the plasma membrane. EPAC2 also interacts with NBD1 of SUR1, being released by cAMP [35]. Such locally released EPAC2 induces the release of Rim2 from the α1.2 Ca L subunit.
The local Ca 2+ influx within Ca L ensures EPAC2 binding to Rim2, and subsequent interaction with another Ca 2+ sensor termed Piccolo. The heterotrimeric complex then interacts with Rab3A and enables IGV exocytosis.
Interestingly, all necessary components of the PKA pathway were identified in the mitochondrial matrix, including sAC, PDE2A2 [132], and also PKA [133]. However, we may also speculate that some proteins can be phosphorylated by cytosolic PKA or by its fraction attached to OMM prior to their import to the mitochondrial matrix. There was also a consensus that cAMP cannot freely diffuse to the matrix [132]. Thus cAMP in the mitochondrial matrix may act as an independent pool [134,135]. Its source is the matrix-located soluble adenylate cyclase sAC, which is activated by bicarbonate and Ca 2+ [136,137]. Since CO 2 is increasingly released when the Krebs cycle turnover increases upon GSIS, the matrix localized mtPKA can be activated in this way [138]. In any case, OXPHOS is facilitated in mitochondria of numerous tissues via phosphorylation of Complex I NDUFS4 subunit (facilitating its Hsp70-mediated import), Complex IV COXIV-1 subunit (preventing its inhibition by ATP) [139] as well as via IF1, enhancing ATP synthesis by disabling the inhibitory binding of phosphorylated IF1 dimers to the ATP synthase [140]. A link to redox homeostasis can be viewed in the observed release of the PKA catalytic subunits by the increased ROS [141,142]. Thus mtPKA can act in parallel to the cytosolic PKA signaling initiated by GPR40 and GLPR or GIPR receptors. PKA targeting of at least IF1, and probably also of Complex I and Complex IV, should contribute to the amplification of insulin secretion by FAs or incretins.
The G protein Gαq/11 initiates signaling through the phospholipase C (PLC-)mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate into DAG and inositol triphosphate IP3 [110]. The main effector of DAG is protein kinase C (PKC), which is activated by DAG. One of the effectors of IP3 is the IP3 receptor (IP3R; subtypes IP3R1, IP3R2 and IP3R3), which is another important Ca 2+ channel residing on ER membranes in β-cells [143]. Similarly to the EPAC2-RyR route of Ca 2+ release from ER Ca 2+ , the opening of this channel amplifies the primary Ca L mediated Ca 2+ signaling for insulin release. PKC contributes to the plasma membrane depolarization, while activating TRPM4 and TRPM5 [144]. Besides the canonical plasma membrane effects, PKC and downstream ERK1/2 signaling stimulates OXPHOS, hence mitochondrial ATP synthesis [145].

GSIS amplification by incretins GLP-1 and GIP
Glucagon-like peptide 1 (GLP-1) and gastric inhibitory polypeptide (GIP) have a prominent impact among other peptides belonging to incretins [107][108][109]. Oral glucose administration provides a higher insulin secretion response than when administered parenterally [146]. This surplus of potentiation of insulin secretion appears to be about equally ascribed to GLP-1 and GIP [147]. Indeed, diminished insulin secretion response to oral glucose was observed in GLP-1 knock out mice [148,149] and was even more decreased in double knockout mice (GLP-1 plus GIP) [149].
Incretin-cAMP signaling amplifies GSIS by both PKA-dependent and EPAC2Adependent pathways. As described above, the EPAC2 pathway is partially dependent on the Ca L opening, and the PKA pathway enables synergy among actions of K ATP , Ca L , and Kv channels, leading again to a more effective Ca L opening. This knowledge complies with the traditional view, considering that the incretin signaling does not stimulate insulin release in the low glucose conditions [150,151]. The GLP1R-cAMP-EPAC2-TRPM2 pathway was suggested to be one of the major routes [106].
GLP-1 is secreted by enteroendocrine L-cells, residing predominantly in the distal ileum and colon. Secretion is initiated by postprandial stimuli, i.e. by glucose, Redox Signaling is Essential for Insulin Secretion DOI: http://dx.doi.org/10.5772/intechopen.94312 fatty acids, or lipids, as well as proteins [152,153]. Only 10 to 15% of active GLP-1 likely reaches the pancreas via the circulation [154]. Thus concentrations of biologically active GLP-1 in human plasma at fasting account for about 2 pmol/l and maximum10 pmol/l postprandially [155], peaking 30 to 60 min after a carbohydrate or protein intake and 120 min after ingestion of lipids [156]. The most efficient truncated variants are GLP-1  and variant GLP-1 (7-36amide) [152]. The latter is ~80% abundant in humans [157]. Note that full peptide GLP-1  is much less efficient in GSIS potentiation [150,151]. Moreover, paracrine GLP-1 signaling acts among the different types of PI cells [150], similarly to the paracrine and endocrine secretion of other hormones. On the systemic level, central control by the brain and nervous system, including GLP-1 secretion in the nucleus tractus solitarii of the brainstem [152], further provides an indispensable top level of regulation for the insulin secretion. GLP-1 effects related to β-cell proliferation or apoptosis are beyond the scope of this review.
The PKA pathway provides a surplus intracellular Ca 2+ above that of the net GSIS without any receptor stimulation. This is ensured by phosphorylation-induced closing among the population of K ATP , stimulation of Ca L opening, and closing of Kv channels [165]. The latter prolongs Ca 2+ stimulation of IGV exocytosis and hence may also potentiate the 2nd phase of GSIS. In parallel, PKA engages snapin interaction with IGVs, reportedly potentiating the 1st GSIS phase [123,124]. Simultaneously, the EPAC2 pathway promotes Ca 2+ -induced RyR-mediated Ca 2+ release from ER, which must be, however, initiated by the ongoing Ca L opening [163]. The EPAC2 pathway also facilitates docking and priming of IGVs by promoting Rab3A interaction with Rim2a [131] and hypothetically interaction of EPAC2-Rim2-Picollo trimers with Rab3A, enabling IGV exocytosis [152]. Stimulation of GLP1R biased downstream via stimulation of Gαq/11 also contributes by a surplus to intracellular Ca 2+ , while inducing the IP3R-mediated Ca 2+ release from ER.
When GLP1 effects were simulated and IGV kinetics was monitored using total internal reflection fluorescence microscopy, cAMP and 8-Br-cAMP were found to increase the frequency of fusion events, i.e. IGV fusion with the plasma membrane in both phases of GSIS [25]. EPAC2A was found to interacts also with a small G protein Rap1, affecting its conformation so to release the catalytic region, which subsequently binds and thus activates another G protein Rap113. In EPAC2A knockout mice, most of the potentiation of the 1st GSIS phase vanished [25]. Thus speculatively, the 2nd phase amplification can be due to the PKA pathway.

Mechanism of insulin secretion stimulated by branched-chain keto-acids
Postprandial response by insulin secretion is also given by substances other than glucose. These substances, which induce the secretion of insulin, are termed secretagogues in general. One important type of secretagogues is branched-chain keto-acids (BCKAs), metabolites of branched-chain amino acids (BCAAs) (Figure 2). We found that the alternative to the NOX4-mediated redox signaling exists for some other insulin secretagogues, particularly for BCKAs [1]. For the redox signaling in this case, the mitochondrial redox signaling replaced that one originating from NOX4. Thus we demonstrated that H 2 O 2 signaling originating from mitochondria is essentially required for insulin secretion stimulated by BCAAs metabolized onto BCKAs, such as 2-ketoisocaproate (KIC; also termed 2-oxoisocaproate, OIC; leucine metabolite), 2-ketoisovalerate (KIV; valine metabolite) and 2-ketomethylvalerate (KMV; isoleucine metabolite) [166,167]. This mechanism was evidenced by the effects of mitochondrial-matrix-targeted antioxidant SkQ1. We observed that SkQ1 did not affect GSIS in INS-1E cells, but completely inhibited insulin secretion stimulated by KIC [1]. Thus the NOX4 source of H 2 O 2 cannot be efficiently inhibited by SkQ1 located within the inner phospholipid leaflet of the inner mitochondrial membrane, whereas the redox signaling originating from mitochondrion must be blocked.
Metabolism of BCKAs begins in the mitochondrial matrix by the reaction of the BCKA dehydrogenase complex (BCKDH), since there is no branched-chain amino acid aminotransferase (BCAT) in the cell cytosol [168]. BCKDH forms isovaleryl-CoA, isobutyryl-Co and methyl-isobutyryl-CoA from KIC, KIV and KMV, respectively. This is followed by a series of reactions resembling β-oxidation of fatty acids. This series, as well as FA β-oxidation, elevates formation of superoxide in the mitochondrial matrix by several ways. The major way is due to the reoxidation of the BCKDH co-factor FADH 2 by the electron-transfer flavoprotein (ETF), the one electron carrier. Two electrons from the two ETF molecules are accepted by the electron-transfer flavoprotein: ubiquinone oxidoreductase (ETF:QOR) [169]. Redox Signaling is Essential for Insulin Secretion DOI: http://dx.doi.org /10.5772/intechopen.94312 ETF:QOR reaction is coupled to ubiquinone (Q ) oxidation to ubiquinol (QH 2 ). This effectively competes with the Complex I reaction of the respiratory chain, also providing Q oxidation to QH 2 driven by NADH. The electron transfer within the Complex I is thus effectively retarded and this results in a higher superoxide formation. Superoxide is then most probably increasingly formed at the I Q site (i.e. at the proximity of the Q-binding site), similarly as due to the reverse electron transfer.
Alternatively, the Complex I electron transfer is retarded upon the acetyl-CoA entry (propionyl-CoA entry for KIV; through methylmalonyl and Succinyl-CoA) into the Krebs cycle. Also, acetoacetate influences redox homeostasis, as one of the final products of leucine metabolism. After superoxide conversion to the elevated H 2 O 2 in the mitochondrial matrix, the H 2 O 2 is elevated in the cytosol and thus represents the mitochondrial retrograde redox signaling. Its target could be again K ATP (or K ATP and TRPM2) which would depolarize the plasma membrane due to this redox signaling ongoing in parallel with the elevated ATP due to concomitantly enhanced OXPHOS.
The BCAA oxidation involves the following sequence of reactions: isovaleryl-CoA dehydrogenase (IVD), methylcrotonyl-CoA carboxylase (MCC), methylglutoconyl-CoA hydratase (MGCoAH) and 3-hydroxy-3-methylglutaryl-CoA lyase (HMGCoAL). The end-products are acetyl-CoA and acetoacetate. Similarly, as for pyruvate metabolism via PDH, the common end-product acetyl-CoA drives the Krebs cycle. This may also increase mitochondrial superoxide formation. Acetyl-CoA is linked to the above acyl-CoA dehydrogenase reaction by the reaction of the ETF:QOR, using ubiquinone (CoQ or Q ) to oxidize it to ubiquinol QH. Also, the ETF:QOR itself may produce superoxide.
In summary, independently of the molecular mechanism, BCAA metabolism leads to the increased mitochondrial superoxide formation. After conversion to H 2 O 2 by the matrix MnSOD and the intermembrane space CuZnSOD, the ongoing H 2 O 2 efflux from mitochondria can be regarded as redox signaling. We have clearly demonstrated that the absence of such redox signaling, for example, in the presence of the mitochondrial matrix-targeted antioxidants SkQ1 leads to a blockage of insulin secretion, which is otherwise stimulated with BCKAs [1]. Likewise, the silencing of BCKDH led to the inhibition of insulin secretion stimulated with BCKAs.

Mechanism of fatty acid-stimulated insulin secretion
Fatty acids (FAs) appear in pancreatic islet capillaries either bound to albumin or being part of postprandial chylomicrons resulting from dietary fat lipids. FA pool of lipoproteins can be also considered. The dietary fat lipids are rich in triglycerides, which are cleaved locally in pancreatic islet capillaries by lipoprotein lipase secreted by β-cells. Resulting 2-monoacylglycerol (2MAG) and long chain FAs [170][171][172][173] stimulate each own two receptors GPR119 [157] and GPR40/FFA1, respectively. Therefore, fatty acid-stimulated insulin secretion (FASIS) could be defined as the net insulin secretion induced at the low glucose concentration, which itself does not stimulate insulin secretion. It is still controversial, whether such a net FASIS exists, since some previous reports observed that glucose should always be present for fatty acid to induce insulin secretion response. In contrast, the other reports described FASIS at 3 mM glucose, but not at zero glucose. Physiologically, postprandial responses should be due to all secretagogues resulting from major saccharide, fat and protein components. FASIS may dominate late responses upon feeding by fatty meal or an experimental high-fat diet [174].
Theoretically, FASIS must concern with the two components (Figure 3). The first one should depend on metabolism and the second one should rely on the stimulation of the metabotropic receptor GPR40. In vivo, FASIS-GPR40 axis is paralleled by a portion of insulin secretion stimulated via another metabotropic receptor, GPR119, to which monoacylglycerol (MAG) binds as the second major component of triglycerides. The metabolic component undoubtedly involves fatty acid β-oxidation, providing both ATP from the elevated OXPHOS and H 2 O 2 from the enhanced superoxide formation by the respiratory chain and ETF:QOR, similarly as for BCKAs [175]. This component is thus directly dependent on K ATP , since it leads to its closure and to the canonical downstream events identical to those during GSIS. Redox Signaling is Essential for Insulin Secretion DOI: http://dx.doi.org /10.5772/intechopen.94312 The receptor component of FASIS may be at least partly K ATP -independent and even Ca L -independent, hence may partly proceed independently of high glucose. In other words, FASIS at low glucose is theoretically possible. The major pathway downstream of GPR40 relies on Gαq/11, which induces PLC-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate into DAG and IP3 [110,111]. The latter would amplify the primary Ca L -mediated Ca 2+ signaling for the insulin release by mediating Ca 2+ release from ER via the Ca 2+ channel of the IP3 receptor [143]. However, this would happen provided that some basal Ca L would be initiated by the metabolic component, i.e. due to partial fatty acid metabolism by β-oxidation followed by H 2 O 2 plus ATP elevations. Also, PKC could be activated downstream of GPR40 as being the main effector of DAG. The PKC pathway could increase the extent of plasma membrane depolarization since it activates TRPM4 and TRPM5 [144]. Moreover, this may act at low glucose, again providing that the initial triggering is ensured by the metabolic component, and so that certain basal H 2 O 2 plus ATP elevations exist, leading to the K ATP closure. Also, another route downstream of GPR40 would involve the Gαq/11-PLC-TRPC-induced Ca 2+ efflux from ER [176]. As mentioned above, the TRPC1-Orai1-STIM1 complex was demonstrated to act during GSIS, while contributing to Ca 2+ oscillations [46,83,84]. The action of such a complex has yet to be studied during experimental FASIS as well as its dependence on Ca L .
Also, biased (promiscuous) pathways of GPR40, i.e., those involving Gα S -cAMP initiation of information signaling may exist and contribute to a certain extent to FASIS. Both downstream pathways of GPR40-Gα S -cAMP stimulation, i.e. the PKA and EPAC2 pathway, could target components of IGV interactions with the plasma membrane, hence being independent of Ca L . These speculations await experimental evidence.
Our in vitro and in vivo experiments with mice (unpublished) demonstrated that approximately 2/3 of the GPR40 response (amplitude of insulin secretion) is given by the amplifying mechanism due to the mitochondrial phospholipase iPLA2γ/PNPLA8 [175]. This phospholipase cleaves both saturated and unsaturated FAs from the phospholipids of mitochondrial membranes. The cleaved free FAs subsequently diffuse up to the plasma membrane, where they activate GPR40. Moreover, the phospholipase iPLA2γ is directly activated by the elevated H 2 O 2 in the mitochondrial matrix. The reader may remain that this is just the FA β-oxidation, which via the increased superoxide formation, due to the function of ETF:QOR and respiratory chain, produces H 2 O 2 , while the concomitant OXPHOS provides elevated ATP. As a result, the sufficient plasma membrane depolarization is enabled. The proof of co-existence of the GPR40 receptor component and metabolic component of FASIS is suggested by the experiments when FASIS in iPLA2γ knockout mice or in its isolated islets yielded only ~30% insulin secretion peak in the 1st phase when compared to wt mice (Holendová B., Jabůrek M, et al., unpublished). Incidentally, a similar portion remains when GW1100 antagonist of GPR40 was applied. These results show that in parallel with the GPR40 pathway, a 1/3 portion of FASIS still results from FA β-oxidation, having a similar mechanism as described for ketoacids. The abolished FASIS in the iPLA2γ knockout mice then supports the existence of such an acute mechanism in vivo, when GPR40 is supplied with mitochondrial fatty acids.

Redox relay as a hypothetical carrier for redox signaling
It has been established that the content of glutathione (GSH) is rather low in pancreatic β-cells [177][178][179][180], in contrast to the content of thioredoxins and  [181,182], peroxiredoxins and other proteins capable of redox relay. Therefore, these proteins are able to conduct and spread the redox signals [183,184]. From this point of view, the pancreatic β-cell appears to be a wellintegrated redox system.

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Redox signal spreading may be accomplished either by the direct diffusion of H 2 O 2 or may be facilitated by the specialized proteins. Redox signals can be traced experimentally as instantly oxidatively modified cysteine residues, which are spread via different sets of proteins in different tissues. However, one may consider their majority as passive targets. For the case of NOX4 residing in the proximity of K ATP , undoubtedly, the direct diffusion of H 2 O 2 would be sufficient. Nevertheless for more distant NOX4 molecules, this would be difficult. Also, for mitochondrial redox signaling towards targets residing in the plasma membrane, a distance over 500 nm must be overcome. Redox signal across such high distances could be conducted through the action of thiol-based proteins capable of redox relay to the target, such as peroxiredoxins (regenerated via thioredoxins and glutaredoxins). The relay would provide a common redox signal transfer. It is yet to be established whether a redox relay exists via an array of peroxiredoxin oligomers.