**3. Mechanisms of the 1st phase of GSIS**

GSIS has been consensually described to involve a so-called triggering mechanism accompanied by amplifying mechanism(s) [7, 12, 30–36]. The triggering is exclusively dependent on the KATP closure and attaining plasma membrane depolarization up to −50 mV. The latter is achieved in a synergy of KATP with other ion channels. The amplifying mechanisms are given by metabolism or stem from the action of incretins and other hormones. Also, mechanisms concerning other secretagogues, such as branched chain ketoacids (BCKAs) and fatty acids (FAs), were considered as merely amplifying. Nevertheless, we will show below the ambiguity of such a classification. The amplifying mechanisms originate from an incremental increase in Ca2+ elevations, not existing within the canonical "triggering" mechanism. Alternatively, they stem from facilitation via numerous proteins of the exocytotic machinery localized either on the IGV or plasma membranes. Therefore, some of these types of events might be Ca2+ independent and hence may also proceed at low glucose concentrations.

The traditional explanation of the triggering mechanism of GSIS relied exclusively on the ATP elevation (or elevation of the ATP/ADP ratio) in the cytosol of β-cells. Sole elevated ATP was considered to be sufficient for the KATP closing [30–33]. Any additional requirement for a parallel redox signaling was not considered, despite the findings that reactive oxygen species (ROS) have been implicated in insulin secretion. This concerned with at least ROS of mitochondrial origin [37], or resulting from mono-oleoyl-glycerol addition [38]. The blockage of PPP, that decreased insulin secretion, also shifted redox homeostasis [39]. An unspecified link of GSIS with the externally added H2O2 was reported, besides antioxidant effects at decreased glutathione by diethylmaleate [40]. Previously, also an unidentified isoform of NADPH oxidase was implicated in GSIS, since an antisense p47PHOX oligonucleotide [41] or an unspecific NOX inhibitors attenuated GSIS [38, 42, 43].

Recently, we have provided the evidence that the elevated OXPHOS is insufficient to initiate GSIS, despite the increased ATP levels and the elevated ATP/ ADP ratio at the peri-plasma- membrane space [1]. We demonstrated that NOX4 is fundamentally required for GSIS [1]. In model rat pancreatic β-cells (INS-1E cells) with silenced NOX4 or in full NOX4 knockout (NOX4KO) mice and in mice with NOX4 knockout, specifically in pancreatic β-cells (NOX4βKO mice), the 1st phase of GSIS was largely blocked [1]. In both studied NOX4 KO mice strains and in their isolated PIs, the 1st phase of GSIS was abolished with NOX4 ablation, while in PIs, either overexpression of NOX4 (achieved at least in the peripheral spheroid layer of islets) or additions of H2O2 rescued this 1st phase. No effects were found in NOX2 KO mice, although NOX2 has been previously implicated to play an antagonistic role for redox homeostasis [44].

Moreover, using a patch-clamp of INS-1E cells, we demonstrated that the KATP closure is possible only when NOX4 is intact in INS-1E cells. After showing the wellknown closure of KATP induced by high glucose concentration in cells transfected with scrambled siRNA, we observed no glucose-induced KATP closure in INS-1E cells silenced for NOX4 [1]. These experiments supported the model, in which KATP integrates metabolic and redox homeostasis and acts as a logical summation for which both elevated ATP plus elevated H2O2 exclusively lead to a triggering of GSIS (**Figure 1**). However, since without cation fluxes provided by nonspecific cation channels a threshold depolarization of −50 mV cannot be achieved, despite 100% KATP ensemble being closed [45, 46], we may also hypothesize that H2O2 alternatively or in parallel activates the TRPM2 channel [2], known to contain redox-sensitive Met residue [47].

Thus our results set a new paradigm for GSIS, since it had never been considered that the sole ATP increase is insufficient for GSIS and is insufficient particularly for the closing of KATP; as well as it had never been considered that any redox signaling might essentially participate in GSIS.

In further work, we also demonstrated that the redox signaling upon GSIS is provided by elevations of cytosolic H2O2, whereas ROS in the mitochondrial matrix (both H2O2 and superoxide release) are diminished due to the enhanced operation

**37**

*Redox Signaling is Essential for Insulin Secretion DOI: http://dx.doi.org/10.5772/intechopen.94312*

dependent (KCa) K+

however, again activated in the next CaL-KV cycle.

the impaired 1st phase of GSIS [63].

malate redox shuttle.

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 H2O2 directly [50, 51], its reaction results in an increase of H2O2 release into the cell cytosol [1]. Finally, the elevated H2O2, 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 CaL opening, followed by the Ca2+ 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 CaL opening, followed by the opening of voltage-dependent channels (KV) in rodents [52] or calcium-

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 [59–62]. 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 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 Ca2+ 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/

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.

#### *Redox Signaling is Essential for Insulin Secretion DOI: http://dx.doi.org/10.5772/intechopen.94312*

*Type 2 Diabetes - From Pathophysiology to Cyber Systems*

also proceed at low glucose concentrations.

role for redox homeostasis [44].

redox-sensitive Met residue [47].

might essentially participate in GSIS.

ion channels. The amplifying mechanisms are given by metabolism or stem from the action of incretins and other hormones. Also, mechanisms concerning other secretagogues, such as branched chain ketoacids (BCKAs) and fatty acids (FAs), were considered as merely amplifying. Nevertheless, we will show below the ambiguity of such a classification. The amplifying mechanisms originate from an incremental increase in Ca2+ elevations, not existing within the canonical "triggering" mechanism. Alternatively, they stem from facilitation via numerous proteins of the exocytotic machinery localized either on the IGV or plasma membranes. Therefore, some of these types of events might be Ca2+ independent and hence may

The traditional explanation of the triggering mechanism of GSIS relied exclusively on the ATP elevation (or elevation of the ATP/ADP ratio) in the cytosol of β-cells. Sole elevated ATP was considered to be sufficient for the KATP closing [30–33]. Any additional requirement for a parallel redox signaling was not considered, despite the findings that reactive oxygen species (ROS) have been implicated in insulin secretion. This concerned with at least ROS of mitochondrial origin [37], or resulting from mono-oleoyl-glycerol addition [38]. The blockage of PPP, that decreased insulin secretion, also shifted redox homeostasis [39]. An unspecified link of GSIS with the externally added H2O2 was reported, besides antioxidant effects at decreased glutathione by diethylmaleate [40]. Previously, also an unidentified isoform of NADPH oxidase was implicated in GSIS, since an antisense p47PHOX oligonucleotide [41] or an unspecific NOX inhibitors attenuated GSIS [38, 42, 43]. Recently, we have provided the evidence that the elevated OXPHOS is insufficient to initiate GSIS, despite the increased ATP levels and the elevated ATP/ ADP ratio at the peri-plasma- membrane space [1]. We demonstrated that NOX4 is fundamentally required for GSIS [1]. In model rat pancreatic β-cells (INS-1E cells) with silenced NOX4 or in full NOX4 knockout (NOX4KO) mice and in mice with NOX4 knockout, specifically in pancreatic β-cells (NOX4βKO mice), the 1st phase of GSIS was largely blocked [1]. In both studied NOX4 KO mice strains and in their isolated PIs, the 1st phase of GSIS was abolished with NOX4 ablation, while in PIs, either overexpression of NOX4 (achieved at least in the peripheral spheroid layer of islets) or additions of H2O2 rescued this 1st phase. No effects were found in NOX2 KO mice, although NOX2 has been previously implicated to play an antagonistic

Moreover, using a patch-clamp of INS-1E cells, we demonstrated that the KATP closure is possible only when NOX4 is intact in INS-1E cells. After showing the wellknown closure of KATP induced by high glucose concentration in cells transfected with scrambled siRNA, we observed no glucose-induced KATP closure in INS-1E cells silenced for NOX4 [1]. These experiments supported the model, in which KATP integrates metabolic and redox homeostasis and acts as a logical summation for which both elevated ATP plus elevated H2O2 exclusively lead to a triggering of GSIS (**Figure 1**). However, since without cation fluxes provided by nonspecific cation channels a threshold depolarization of −50 mV cannot be achieved, despite 100% KATP ensemble being closed [45, 46], we may also hypothesize that H2O2 alternatively or in parallel activates the TRPM2 channel [2], known to contain

Thus our results set a new paradigm for GSIS, since it had never been considered that the sole ATP increase is insufficient for GSIS and is insufficient particularly for the closing of KATP; as well as it had never been considered that any redox signaling

In further work, we also demonstrated that the redox signaling upon GSIS is provided by elevations of cytosolic H2O2, whereas ROS in the mitochondrial matrix (both H2O2 and superoxide release) are diminished due to the enhanced operation

**36**

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 H2O2 directly [50, 51], its reaction results in an increase of H2O2 release into the cell cytosol [1]. Finally, the elevated H2O2, 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 CaL opening, followed by the Ca2+ 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 CaL opening, followed by the opening of voltage-dependent channels (KV) in rodents [52] or calciumdependent (KCa) K+ -channels in humans. Their action deactivates CaL, which are, however, again activated in the next CaL-KV 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 [59–62]. 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 Ca2+ 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.