An agreed-upon consensus model of glucose-stimulated insulin secretion from healthy β-cells is essential for understanding diabetes pathophysiology. Since the discovery of the KATP channel in 1984, an oxidative phosphorylation (OxPhos)–driven rise in ATP has been assumed to close KATP channels to initiate insulin secretion. This model lacks any evidence, genetic or otherwise, that mitochondria possess the bioenergetics to raise the ATP/ADP ratio to the triggering threshold, and conflicts with genetic evidence demonstrating that OxPhos is dispensable for insulin secretion. It also conflates the stoichiometric yield of OxPhos with thermodynamics, and overestimates OxPhos by failing to account for established features of β-cell metabolism, such as leak, anaplerosis, cataplerosis, and NADPH production that subtract from the efficiency of mitochondrial ATP production. We have proposed an alternative model, based on the spatial and bioenergetic specializations of β-cell metabolism, in which glycolysis initiates insulin secretion. The evidence for this model includes that 1) glycolysis has high control strength over insulin secretion; 2) glycolysis is active at the correct time to explain KATP channel closure; 3) plasma membrane–associated glycolytic enzymes control KATP channels; 4) pyruvate kinase has favorable bioenergetics, relative to OxPhos, for raising ATP/ADP; and 5) OxPhos stalls before membrane depolarization and increases after. Although several key experiments remain to evaluate this model, the 1984 model is based purely on circumstantial evidence and must be rescued by causal, mechanistic experiments if it is to endure.

The importance of glucose metabolism for triggering insulin secretion was well established by the 1960s, when glucokinase was identified as the β-cell glucose sensor. The requirement for electrical excitability followed shortly after, and the 1984 discovery of the β-cell KATP channel provided a metabolic actuator for plasma membrane electrical activity that is sensitive to the ATP/ADP ratio and initiates Ca2+ influx (1). In the subsequent four decades, mitochondrial oxidative phosphorylation (OxPhos) has been positioned as the exclusive, intervening step that raises cytosolic ATP/ADP (ATP/ADPc) to close KATP channels and initiate insulin secretion (1). While the roles of glucokinase and KATP channels have been codified by gain- and loss-of-function mutations in humans (causing either hypoglycemia or neonatal diabetes), is there anything more than circumstantial, correlative evidence for the hypothesis that a glucose-dependent increase in mitochondrial ATP production closes KATP channels? As reviewed by Rutter and Sweet (2), the answer is no, whereas causative mechanistic experiments suggest that glycolysis has preferential, if not exclusive, control over KATP channel closure.

β-Cell glucose is almost completely oxidized in the mitochondria due to the absence of pyruvate/lactate transporters. As such, often-cited evidence for the canonical model is that mitochondrial poisons (rotenone, oligomycin, etc.) prevent KATP channel closure (3). Rutter and Sweet (2) point out a flaw in this argument: since glycolytic and mitochondrial metabolism are functionally entangled, perturbations of one will impact the other. For instance, poisoning mitochondria hinders not only glycolysis but also other nonoxidative mitochondrial fluxes (e.g., the phosphoenolpyruvate [PEP] cycle, which contributes glycolytic metabolites). However, the tight coupling between glycolysis and mitochondria has been used to argue that the number of ATPs produced by glycolysis is dwarfed by OxPhos in order to support the canonical view.

We do not dispute in β-cells that 1) glucose elevation increases OxPhos, 2) mitochondria are essential to stimulus-secretion coupling, and 3) mitochondrial health and dysfunction in diabetes should be aggressively studied. We simply argue that an OxPhos-independent increase in ATP/ADP, driven by glycolysis, closes KATP channels. This postulate is supported by recent evidence demonstrating that deletion of the respiratory chain complexes severely impairs OxPhos but does not impact insulin secretion (4). What, then, is the relevant mechanism? We propose that the spatial privilege and favorable bioenergetics of the pyruvate kinase (PK) reaction are what matter for KATP closure (Fig. 1).

Figure 1

Synopsis of arguments for and against the canonical model of glucose-stimulated insulin secretion. Figure created with BioRender.com.

Figure 1

Synopsis of arguments for and against the canonical model of glucose-stimulated insulin secretion. Figure created with BioRender.com.

Close modal

The primary evidence in support of the canonical model is that a rise in NAD(P)H, ATP/ADPc, and O2 consumption precedes Ca2+ influx (5–7). This evidence not only is corollary but also is commonly obtained by rescuing ATP/ADPc from a fuel-starved state to a fuel-saturated one. This situation never occurs in vivo. In both the fed and fasted states, insulin secretion oscillates with a 5- to 10-min period; glucose increases the pulse amplitude rather than “switching on” secretion (8,9). When islet metabolic signaling is observed during such physiological oscillations, multiple approaches suggest that OxPhos turns off before KATP channels close and reactivates after membrane depolarization and Ca2+ influx (5,6,10,11). While these temporal experiments are correlative, not causal, they led to our search for an alternative ATP generator that is fuel dependent but active at the time KATP channels close.

In the last step of glycolysis, PK converts ADP and PEP to ATP and pyruvate. There are three PK isoforms in β-cells: PKm1 is constitutively active, while PKm2 and PKL require allosteric activation by fructose 1,6-bisphosphate (FBP), which is produced earlier in glycolysis by the phosphofructokinase reaction. Phosphofructokinase has strong control over Ca2+ oscillations (12), and PKm2 activity peaks at the time KATP channels close (13). PK activation was found to increase the frequency of Ca2+ oscillations, and subsequently, recordings from excised patches of human and mouse β-cell plasma membranes demonstrated that the associated PK raises the plasma membrane ATP/ADP ratio (ATP/ADPpm) to close KATP channels (12). β-Cell deletion of PKm1 and PKm2 revealed isoform redundancy for KATP channel regulation, provided that FBP (or a pharmacologic PK activator) was present to activate PKm2 (14). This work also overturns the canonical model by demonstrating PK is essential for KATP channel closure while ATP/ADPc is not. Mitochondrial PEP, generated by mitochondrial PCK2 in the PEP cycle, was found to accelerate glucose-dependent KATP channel closure but is not essential (14,15). Finally, in human and mouse β-cells, PK is part of a plasma membrane–associated glycolytic metabolon that regulates KATP channel opening and closure via ATP/ADPpm (16) (Fig. 2). These data support earlier biochemical studies, including in MIN6 cells, showing that KATP channel subunits interact with the enzymes of upper and lower glycolysis (17–19).

Figure 2

The spatial bioenergetic model of β-cell KATP regulation by PK. A: When glucose is elevated, glycolytic and mitochondrially derived PEP is delivered to PK, which is part of a KATP-containing glycolytic metabolon within plasma membrane microdomains. PEP hydrolysis raises the local ATP/ADP ratio, closing KATP channels, which depolarizes the plasma membrane to activate voltage-gated Ca2+ channels that stimulate insulin release. B: The PEP cycle is a step-up transformer that redistributes the free energy of mitochondrial matrix ATP and GTP into PEP, a high-energy signal that returns to the cytosol. In the first half of the cycle, one ATP molecule and one GTP molecule are consumed to generate a single PEP molecule from pyruvate. This reaction supercharges PEP with approximately twice the bioenergetic potential of ATP. The second half of the PEP cycle uses the energy released from cytosolic PEP hydrolysis to deprive mitochondria of ADP, turning off OxPhos while at the same time raising the ATP/ADP ratio to the KATP-triggering threshold. Figure created with BioRender.com.

Figure 2

The spatial bioenergetic model of β-cell KATP regulation by PK. A: When glucose is elevated, glycolytic and mitochondrially derived PEP is delivered to PK, which is part of a KATP-containing glycolytic metabolon within plasma membrane microdomains. PEP hydrolysis raises the local ATP/ADP ratio, closing KATP channels, which depolarizes the plasma membrane to activate voltage-gated Ca2+ channels that stimulate insulin release. B: The PEP cycle is a step-up transformer that redistributes the free energy of mitochondrial matrix ATP and GTP into PEP, a high-energy signal that returns to the cytosol. In the first half of the cycle, one ATP molecule and one GTP molecule are consumed to generate a single PEP molecule from pyruvate. This reaction supercharges PEP with approximately twice the bioenergetic potential of ATP. The second half of the PEP cycle uses the energy released from cytosolic PEP hydrolysis to deprive mitochondria of ADP, turning off OxPhos while at the same time raising the ATP/ADP ratio to the KATP-triggering threshold. Figure created with BioRender.com.

Close modal

Alternative explanations for compartmentalized KATP channel closure have been proposed. 1) KATP channels were closed in excised patch experiments by the failure to adjust the pH of the solutions after adding PEP (3); knockout of PK excludes this possibility (14). Importantly, the bath solution pH was adjusted to 7.2 after adding metabolites in all our studies (11,14,16) and in prior studies that demonstrated glycolytic regulation of KATP in cardiac myocytes (17,18,20–22). Taken together, PEP and ADP closed plasma membrane KATP channels in eight of these nine electrophysiology studies (five of five cardiomyocyte studies and three of four β-cell studies, with the exception of Corradi et al. [3]). 2) PEP could directly close KATP channels without its metabolism; knockout of PK also excludes this possibility (14). 3) KATP channels were closed by OxPhos via stray mitochondria associated with excised patches; this possibility was excluded because pyruvate and ADP do not close KATP channels in excised patches, and oligomycin does not prevent KATP channel closure by PK substrates (16). 4) Excised patches are recessed into the pipette, which could exaggerate local KATP channel regulation by membrane-associated glycolytic enzymes. Most likely, this last issue will remain unresolved until experiments conclusively test whether the location of PK is important for KATP channel regulation. A key question is whether ATP must diffuse into the microvilli, which blanket the β-cell plasma membrane (22) and are even smaller than the patch pipette. If KATP channels are located at the microvillus tip, rather than the base, the kinetic limitation on nucleotide diffusion would favor local KATP regulation by glycolysis.

To clarify our position, our published studies have not argued that glycolysis preferentially regulates KATP channels because ATP is unable to diffuse from mitochondria to the plasma membrane, as in the canonical model, just that OxPhos cannot make ATP at the time of KATP closure (due to the underlying bioenergetics, described below) (11). That said, the conclusion of Rutter and Sweet (2) that ATP/ADPc and ATP/ADPpm are similar if not identical should be reevaluated. First, the available experiments used untargeted Perceval sensors (23) that cannot distinguish plasma membrane versus cytosol, or luciferase (24) that detects ATP rather than ATP/ADP. Second, they make the unproven and unlikely assumption that PK does not contribute to the dynamics of ATP/ADPc. Given that mitochondrial PK activity locally inhibits OxPhos (11), it would similarly shape ATP/ADPc. Indeed, it would be surprising if ATP/ADPc and ATP/ADPpm were identical, since the plasma membrane is full of Ca2+-ATPases and Na+/K+-ATPases (25). Until proven otherwise, it remains plausible that mitochondrially derived ATP is inefficient for signaling to KATP channels relative to mitochondrially derived PEP, which has few interactions (limited to enolase, PCK2, and PK) and demonstrably signals to the plasma membrane (14,15).

Another source of confusion is that mice lacking either PKm1 or PKm2 in β-cells have mild phenotypes. For example, β-cell deletion of PKm1 reduces islet PK activity by ∼90% but does not perturb glucose-stimulated Ca2+ oscillations or glucose-stimulated insulin secretion (14). How, then, can Foster et al. (14) claim that PK is essential for KATP closure? To test this hypothesis, the redundancy of PKm1 and PKm2 at elevated glucose concentrations was avoided by using mitochondria (rather than glycolysis) as the sole source of PEP for PK. At low glucose (2 mmol/L), leucine stimulates mitochondria to make PEP from glutamine without making FBP. Mitochondrial PEP can only be utilized by constitutively active PKm1, not by PKm2 or PKL, which both require FBP. Consequently, amino acids do not stimulate KATP closure, Ca2+ influx, and insulin secretion following β-cell deletion of PCK2 (which prevents mitochondrial PEP production) or PKm1 (the only FBP-independent isoform) (14,15). These experiments show that PK is essential for KATP closure and membrane depolarization. The knockouts were not sick—pharmacologic activation of PKm2 (a stand-in for glycolytic FBP) immediately restored the ability of amino acids to close KATP channels in β-cells lacking PKm1 (so does glucose, which explains the mild phenotype of the mice). Importantly, the amino acid–stimulated rise in ATP/ADPc in PKm1-deficient β-cells is comparable to that of controls, yet KATP channels did not close, indicating that ATP/ADPc is insufficient to close KATP channels.

With this new knowledge of KATP channel regulation, it makes sense that a recent report (3) examined KATP channel activity and found no impact of a PKm2 inhibitor (because PKm1 is also present). They also found no impact of a PKm2/L activator on KATP channel activity at low glucose (because there is no PEP) or at high glucose (because FBP is already present). Importantly, PKm2/L activation accelerates Ca2+ oscillations and increases insulin secretion from mouse and human islets (11,14,16). These assays were not performed by Corradi et al. (3). Our experiments (11,14,16) also explain why mitochondrial fuels (e.g., glutamine/leucine, α-ketoisocaproate, and in insulinomas, pyruvate) increase O2 consumption, raise ATP/ADPc, close KATP, and initiate Ca2+ influx and insulin release even when glucose is low. Since mitochondrial PCK2 synthesizes PEP, none of these mitochondrial substrates bypass the PK reaction (14). The newly described PCK2-PK-KATP pathway provides a hypothesis to explain how activating mutations in glutamate dehydrogenase causes hyperinsulinemic hypoglycemia in response to protein-rich meals (even without carbohydrates) (26).

Nature provides redundancy for critical processes, as shown for KATP regulation by PKm1 and PKm2 (14). A fair question, raised by the field, is what happens when both isoforms are deleted in β-cells? Rest assured, the phenotype is impressive, but it was important to first demonstrate that PK is necessary and sufficient for KATP closure in healthy β-cells (i.e., in the absence of hyperglycemia) (11,14,16). That <10% of PKm1 protein can facilitate normal glucose-stimulated insulin secretion (14) supports the argument that metabolic compartmentation is highly effective.

To the extent that the ATP/ADP ratio influences the KATP channel, the biochemical and bioenergetic constraints that determine this ratio are crucial. Our working model is that at rest and following depolarization, OxPhos maintains a nonstimulatory ATP/ADPc ratio. The model considers that β-cell mitochondria are inefficient ATP generators lacking sufficient thermodynamic strength to raise ATP/ADPc to closing thresholds. This oxidative inefficiency together with amplification by the PEP cycle further augments the PK reaction, a reaction that has favorable bioenergetics to raise ATP/ADPpm to KATP-triggering levels.

β-Cell Mitochondria Are Low-Yield ATP Generators

In a highly coupled tissue, like muscle, for every glycolytic substrate (e.g., one-half glucose, glyceraldehyde, or dihydroxyacetone) that is fully oxidized, a mere one ATP comes from the PK reaction compared with the ∼14 ATPs arising from OxPhos, calculated using modern phosphate-to-oxygen ratios (27) (Fig. 3). If this situation were imposed on β-cells, it would be easy to discount the glycolytic contribution in favor of the high OxPhos yield. However, the established metabolic features of β-cells make them different than muscle and diminishes the stoichiometric dominance of OxPhos (28). For instance, up to half of cellular PEP arises from the PEP cycle, which also consumes one ATP molecule and one GTP molecule in the mitochondrial matrix (29–32). Thus, PEP cycling doubles the yield of PK-derived ATP while undermining the OxPhos yield (Fig. 3). Furthermore, β-cell mitochondria are remarkably uncoupled, such that more than half of the proton motive force (PMF) does not contribute to ATP synthesis (30,33). Taken together, the yield of ATP for the relative PK-to-OxPhos ratio is about 1:3.4. In addition, there is substantial evidence for NADPH biosynthesis in β-cells via isoforms of malic enzyme, isocitrate dehydrogenase, and/or nicotinamide nucleotide transhydrogenase (34). Each NADP+ molecule reduced to NADPH by these enzymes prevents oxidation of 1 NADH molecule at a cost of at least 3.7 ATP molecules. This lowers the yield to about 1:2.7 if a single NADPH molecule is generated. The PK-to-OxPhos ratio further increases by the transient diversion of pyruvate from oxidation into enlarging the cytosolic citrate pool (1:1.3) and glutamate pool (1:2) (11,35). Likewise, mitochondrial Ca2+ homeostasis has a large energetic cost, where cycling of one Ca2+ in and out of the mitochondria could have been used to make 0.8 ATP molecules. (Ca2+ activates matrix dehydrogenases by lowering the substrate Km and not by increasing Vmax. In a cycle, this will just change the relative concentrations of metabolites.) Thus, depending on the amount of leak (approaching 100% at peak PMF), PEP cycling, cataplerosis, and Ca2+ cycling, the level of PK-derived ATP approaches or potentially exceeds that of OxPhos (Fig. 3). The mitochondrion-to-PK ratio is likely an underestimate of the PK contribution during the electrically silent phase prior to depolarization. As such, considering stoichiometric yield alone, β-cell OxPhos is an overestimated source of ATP.

Figure 3

Mitochondrial and glycolytic accounting. The number of protons pumped per ATP molecule generated in the cytosol versus mitochondrial matrix for muscle and β-cell mitochondria is shown (left) using modern assumptions (right) during the metabolism of 1/2 glucose. The total number of protons pumped is reduced by proton leak (measured in the different tissues) before calculating the ATP made by OxPhos and transported to the cytosol (Mito ATPc) at the cost of one proton. The ratio of mitochondrially generated ATP (including substrate-level ATP from succinyl CoA synthetase [SCS]) versus PK-generated ATPc is reported for muscle with 10% leak or β-cells with 50% leak by itself, with one PEP cycle, or with one PEP cycle plus one NADPH molecule generated by isocitrate dehydrogenase (ICDH or IDH) or malic enzyme (ME). In addition, the expense of increasing cytosolic glutamate or citrate (per two pyruvate molecules, one through pyruvate dehydrogenase [PDH] and one through pyruvate carboxylase [PC]) is also shown. The final mitochondrion-to-PK (Mito/PK) ratio will be a function of the fractional contributions of the fluxes associated with PEP cycling, NADPH synthesis, cataplerosis, and Ca2+ cycling and may vary between the electrically silent and active phases of secretion. This will lead to an underestimation of the PK contribution and an overestimation of OxPhos during the electrically silent phase when metabolic signaling occurs. The malate-aspartate shuttle was used for muscle and the glycerophosphate shuttle (GPD2) was used for β-cells to transport electrons from GAPDH-generated NADH. The phosphate-to-oxygen ratio is based on the stoichiometry of three ATP molecules per full turn and eight c-subunits/complex V. A lowercase c or m suffix refers to the location, cytosol or mitochondria, respectively, where the cofactor is generated. αKGDH, α-ketoglutarate dehydrogenase; ANT, adenine nucleotide translocator; FADH, flavin adenine dinucleotide; MDH, malate dehydrogenase; MiCU, mitochondrial Ca2+ uniporter; NCLX, mitochondrial Na+/Ca2+ exchanger; NNT, nicotinamide nucleotide transhydrogenase; PEPCK, phosphoenolpyruvate carboxykinase; SDH, succinate dehydrogenase. Figure created with BioRender.com.

Figure 3

Mitochondrial and glycolytic accounting. The number of protons pumped per ATP molecule generated in the cytosol versus mitochondrial matrix for muscle and β-cell mitochondria is shown (left) using modern assumptions (right) during the metabolism of 1/2 glucose. The total number of protons pumped is reduced by proton leak (measured in the different tissues) before calculating the ATP made by OxPhos and transported to the cytosol (Mito ATPc) at the cost of one proton. The ratio of mitochondrially generated ATP (including substrate-level ATP from succinyl CoA synthetase [SCS]) versus PK-generated ATPc is reported for muscle with 10% leak or β-cells with 50% leak by itself, with one PEP cycle, or with one PEP cycle plus one NADPH molecule generated by isocitrate dehydrogenase (ICDH or IDH) or malic enzyme (ME). In addition, the expense of increasing cytosolic glutamate or citrate (per two pyruvate molecules, one through pyruvate dehydrogenase [PDH] and one through pyruvate carboxylase [PC]) is also shown. The final mitochondrion-to-PK (Mito/PK) ratio will be a function of the fractional contributions of the fluxes associated with PEP cycling, NADPH synthesis, cataplerosis, and Ca2+ cycling and may vary between the electrically silent and active phases of secretion. This will lead to an underestimation of the PK contribution and an overestimation of OxPhos during the electrically silent phase when metabolic signaling occurs. The malate-aspartate shuttle was used for muscle and the glycerophosphate shuttle (GPD2) was used for β-cells to transport electrons from GAPDH-generated NADH. The phosphate-to-oxygen ratio is based on the stoichiometry of three ATP molecules per full turn and eight c-subunits/complex V. A lowercase c or m suffix refers to the location, cytosol or mitochondria, respectively, where the cofactor is generated. αKGDH, α-ketoglutarate dehydrogenase; ANT, adenine nucleotide translocator; FADH, flavin adenine dinucleotide; MDH, malate dehydrogenase; MiCU, mitochondrial Ca2+ uniporter; NCLX, mitochondrial Na+/Ca2+ exchanger; NNT, nicotinamide nucleotide transhydrogenase; PEPCK, phosphoenolpyruvate carboxykinase; SDH, succinate dehydrogenase. Figure created with BioRender.com.

Close modal

OxPhos Is Hobbled in β-Cells

Stoichiometric yield arguments have little relevance to the bioenergetics that actually determine the ATP/ADP ratio (27). Regardless of the initial source of ATP, cytosolic ATP is buffered by rapid equilibration with creatine kinase and holds the millimolar ATP concentration nearly constant in the cytosol, while ADP is ∼2–3 orders of magnitude more dilute. As such, ATP/ADPc is dominantly influenced by lowering the small amount of ADP, not by adding more ATP (27,34). As ATP/ADPc gets lower, the energetic requirement becomes steeper (28). Thus, increasing the ATP/ADPc to triggering levels is a function of the bioenergetics (ΔGp) rather than stoichiometric yield arguments (Fig. 1). In this regard, ΔGp is set by the PMF and defines the maximal ATP/ADP that OxPhos can generate. The maximum PMF that β-cell mitochondria reach is an embarrassing −140 to −145 mV compared with muscle’s −180 to −200 mV (Fig. 4A). In addition, when mitochondria are hyperpolarized, they apparently jettison reduced cytochrome c to the cytosol, further impairing the respiratory chain capacity (36). In this regard, β-cell mitochondria are severely disadvantaged by sizable leak currents that lower the maximal PMF and throttle back the ADP-lowering potential (ΔGp).

Figure 4

β-Cell mitochondrial bioenergetics. A: Leak and respiration curves of β-cell and muscle mitochondria (data are from Affourtit and Brand [27]). Oxygen consumption (Jo) and mitochondrial membrane potential (ΔΨm) were measured during modular titration of malonate (respiration) and FCCP (leak) in oligomycin- and nigericin-treated mitochondria. Square markers refer to values of state 3 respiration in both tissues, and circles refer to the states corresponding to B, C, and D. Both ΔΨm and Jo were remarkably smaller for β-cells than for mitochondria. Note that even at low glucose (approximately state 3), >50% of respiration was mitochondrial leak in β-cells. The OxPhos-attributable respiration further declined, moving toward ∼0% during state 4 respiration on the curve while leak increased to 100%. B–D: Hydrodynamic model of glucose sensing in β-cells. B: At low glucose, PEP is produced by low rates of glycolysis (GK and PK) and amino acid (PCK2) metabolism so PK can supply pyruvate for oxidation to generate a proton gradient (Δp ∼130 mV). The combination of intrinsic proton leak and basal ATP turnover maintains a steady supply of ADP such that complex V (ATP synthase) consumes the proton gradient to sustain the ATP/ADP ratio at a subtriggering ratio (ΔGp). C: After glucose is raised but prior to depolarization, glycolysis and the PEP cycle use PK to lower ADP, which raises ΔGp nearer to the KATP-triggering threshold. The hyperpolarized mitochondria augment the PEP cycle, supercharging PEP, which feeds forward to amplify the process. This ADP lowering restricts the ability of OxPhos to make ATP and increases Δp to a state 4-like maximum of ∼145 mV that drives a large ohmic increase in proton leak. D: Following depolarization, plasma membrane ATPases consume ATP to generate ADP, reactivating OxPhos and slightly lowering Δp with proportional reduction in both leak and PEP cycling. CV, complex V/ATP synthase; GK, glucokinase; PC, pyruvate carboxylase. Figure created with BioRender.com.

Figure 4

β-Cell mitochondrial bioenergetics. A: Leak and respiration curves of β-cell and muscle mitochondria (data are from Affourtit and Brand [27]). Oxygen consumption (Jo) and mitochondrial membrane potential (ΔΨm) were measured during modular titration of malonate (respiration) and FCCP (leak) in oligomycin- and nigericin-treated mitochondria. Square markers refer to values of state 3 respiration in both tissues, and circles refer to the states corresponding to B, C, and D. Both ΔΨm and Jo were remarkably smaller for β-cells than for mitochondria. Note that even at low glucose (approximately state 3), >50% of respiration was mitochondrial leak in β-cells. The OxPhos-attributable respiration further declined, moving toward ∼0% during state 4 respiration on the curve while leak increased to 100%. B–D: Hydrodynamic model of glucose sensing in β-cells. B: At low glucose, PEP is produced by low rates of glycolysis (GK and PK) and amino acid (PCK2) metabolism so PK can supply pyruvate for oxidation to generate a proton gradient (Δp ∼130 mV). The combination of intrinsic proton leak and basal ATP turnover maintains a steady supply of ADP such that complex V (ATP synthase) consumes the proton gradient to sustain the ATP/ADP ratio at a subtriggering ratio (ΔGp). C: After glucose is raised but prior to depolarization, glycolysis and the PEP cycle use PK to lower ADP, which raises ΔGp nearer to the KATP-triggering threshold. The hyperpolarized mitochondria augment the PEP cycle, supercharging PEP, which feeds forward to amplify the process. This ADP lowering restricts the ability of OxPhos to make ATP and increases Δp to a state 4-like maximum of ∼145 mV that drives a large ohmic increase in proton leak. D: Following depolarization, plasma membrane ATPases consume ATP to generate ADP, reactivating OxPhos and slightly lowering Δp with proportional reduction in both leak and PEP cycling. CV, complex V/ATP synthase; GK, glucokinase; PC, pyruvate carboxylase. Figure created with BioRender.com.

Close modal

O2 Consumption Does Not Equal OxPhos

The increased islet respiration following a glucose increase is often interpreted by proponents of the canonical model as substrate “pushing” OxPhos to raise the ATP/ADP ratio, but this logic is backwards. Following a sudden increase in glucose, or plasma membrane depolarization, 1) ADP is increased (from increased glucose phosphorylation or other ATP-hydrolyzing work), 2) ATP synthase then makes ATP from ADP, which 3) lowers the PMF, which 4) increases O2 consumption (Fig. 4B–D). That is to say, O2 consumption increases as ATP/ADP levels decrease, not vice versa. In contrast, as the ATP/ADPc ratio approaches the PMF allowed maximum (−145 mV), ATP synthase slows to a stop while mitochondrial leak proportionately increases ohmic proton conductance, increasing O2 consumption (37). At these voltages, O2 consumption correlates positively with leak and inversely with OxPhos and therefore does not support the canonical model.

Modifying Mitochondrial Metabolism Breaks the Canonical Model

The canonical model predicts substrate supply–driven OxPhos increases ATP/ADPc, so perturbations that increase substrate supply, mitochondrial membrane potential (ΔΨm; a close surrogate of PMF), and matrix ATP synthesis, or decrease ATP-consuming pathways, all should enhance insulin secretion. Substantial work directly challenges this view. For instance, acutely closing mitochondrial leaks, reducing cytosolic ATP consumption, decreasing anaplerosis and cataplerosis, increasing NAD+ reduction of pyruvate flux, and increasing matrix substrate–level ATP synthesis all decreased insulin secretion (15,30–33,37,38). Whereas increased mitochondrial leak increased basal insulin secretion, decreased substrate-level matrix ATP synthesis increased insulin secretion (30,31,33,38). Notably, the changes with insulin secretion associated with PEP cycling did not correlate with O2 consumption, ΔΨm, or NAD(P)H reduction (30–32). Each of the above treatments is explainable by changes in PK flux but inversely correlate with OxPhos. As a potential final blow to the canonical model, recent data dismiss the role of the electron transport chain for glucose-stimulated insulin secretion (4).

β-Cell OxPhos Is Not Supply Sided

The canonical model is built on the premise that β-cell mitochondria are regulated by substrate supply, as opposed to the mitochondria of all other tissues, where demand controls OxPhos (27). Demand is generated from work-related hydrolysis of ATP to ADP together with Ca2+, which signals OxPhos to activate (39). Ca2+ also may have a more important role in activating glycolysis to increase the pyruvate supply for anaplerotic, cataplerotic, or oxidative metabolism rather than activating OxPhos per se (40). For demand mitochondria, the ATP/ADPc ratio equilibrium when ATP consumption and production are matched is set near the maximum PMF voltage. An increase in cellular work drops the ATP/ADPc ratio, which increases OxPhos and lowers PMF to activate respiration and explains why we breathe harder with exercise than after drinking a soda. In contrast, a supply regimen presupposes that a glucose-induced increase in pyruvate pushes mitochondria to raise the PMF to increase OxPhos. This model assumes that while at baseline, β-cell mitochondria are deprived of substrate, which lowers the PMF. A prediction of such a model is that increased demand from further lowering the ATP/ADPc ratio (e.g., via mitochondrial uncoupling) could not increase respiration, since, being in a fuel-deficient state, there is no excess fuel to oxidize. Contrary to this canonical prediction, uncoupling β-cell mitochondria even at low glucose dramatically increases respiration, even more than substrate can stimulate it, and formally rules out supply-sided regulation (11). Using canonical reasoning, pharmacologic activation of distal glycolysis (e.g., PK activation) could not increase OxPhos to increase insulin secretion, since glucokinase controls the pyruvate supply. The opposite is observed, namely, PK activation dramatically enhances insulin secretion (11).

There is a third possibility, where mitochondria remain demand regulated while PK is substrate supply regulated, which may explain how glucose is actually sensed. In this model, OxPhos defends the resting ATP/ADPc using the same demand principles common to all mitochondria. However, because the mitochondria are hobbled by substantial proton leak, the equilibrium ATP/ADPc is held lower, below the threshold to close KATP channels. As glucose is increased, PK uses the augmented PEP supply (coming from glucokinase and PCK2) to raise the ATP/ADPpm ratio to KATP channel–triggering levels. This is a testable model that does not require a complete reimagining of mitochondrial function; however, definitive quantitative characterization of these bioenergetic constraints is needed.

Phosphoenolpyruvate Has More Pep

For PK to respond to supply-sided regulation, the reaction must have a higher ΔGp than mitochondria can muster. This has been borne out in permeabilized β-cells where PEP suppresses O2 consumption (11), an effect mimicked by PK activation in intact cells (41). The more favorable energetics of PK are linked to the keto-enol tautomerization of pyruvate, leaving PEP with the largest drop in free energy following hydrolysis of all phosphorylated metabolites in the cell, including ATP (42). This is apparent if one considers two net reactions that comprise the two halves of the PEP cycle (Fig. 2B):

Consequently, two mitochondrial ATP equivalents power the synthesis of one PEP equivalent, leaving it with more favorable bioenergetics than OxPhos to lower ADPc. As such, the PEP cycle acts like a step-up transformer that supercharges the cytosolic ADP–lowering potential of PK (Fig. 2B). This also explains how PEP induces mitochondrial hyperpolarization by ADP privation and generates triggering ATP/ADPpm ratios locally at KATP channels.

PK Controls OxPhos and the ATP/ADPpm Ratio

Glycolysis has been portrayed as a passive conduit with the sole purpose of delivering pyruvate to the mitochondria while generating insignificant ATP. In β-cells, these assumptions are inaccurate and should be replaced with a model where PEP hydrolysis by PK lowers ADP, which closes KATP channels and inhibits OxPhos prior to membrane depolarization. β-Cell mitochondria have evolved to be extremely leaky, a feature that keeps the ATP/ADPc ratio relatively low but increases flexibility to augment PK flux when glucose levels are increased. PK harnesses the free energy of PEP hydrolysis to deprive mitochondria and KATP channels of ADP. ADP privation in hyperpolarized mitochondria feeds forward to induce PEP cycling to further amplify ADP lowering. Importantly, in this model OxPhos is still extremely important and mitochondria still retain responsiveness to ADP following plasma membrane depolarization to support rapid ATP synthesis, but OxPhos cannot affect KATP channel closure.

In the end, the 1984 model lacks mechanistic support and should be replaced whether or not the details of our spatial and bioenergetic arguments prove correct. Given the 1960s observations that glycolysis has strong control strength over insulin secretion, our hypothesized model of glycolytic KATP channel regulation is plausible and offers better paradigms to understand and treat β-cell dysfunction. Nonetheless, important experiments remain to be performed:

  • 1.

    While we have provided evidence that PK is essential for KATP channel closure, we have not yet tested whether the location of glycolysis on the plasma membrane (or other organelles) is necessary for efficient β-cell stimulus-secretion coupling. Notably, the location of PK may not matter if PK outperforms OxPhos bioenergetically; either argument overturns the canonical model.

  • 2.

    There is a KATP channel–independent or amplifying effect of PK, revealed by exocytosis experiments that included PK activators in the patch pipette or experiments conducted in intact islets treated with diazoxide and KCl (11). Consistent with this finding, PEP plus ADP, but not pyruvate and ADP, stimulate biphasic insulin release in permeabilized islets (even in the presence of rotenone) (43). While the mechanistic basis of this amplifying effect remains to be clarified, these findings do not argue against KATP channel regulation by PK, as claimed by Rutter and Sweet (2).

  • 3.

    Our working model is that β-cell mitochondria evolved to be incapable of raising the ATP/ADPc ratio sufficiently to close KATP channels via OxPhos alone, and play a supportive role that expands after depolarization. PK, fueled by glycolysis and the PEP cycle, is responsive to changes in nutrients and has favorable thermodynamics to close KATP channels as well as to deprive mitochondria of ADP. While gaps in this model remain, quantitative bioenergetic studies are underway.

See accompanying articles, pp. 844 and 849.

This article is featured in a podcast available at diabetesjournals.org/diabetes/pages/diabetesbio.

Funding. This work received support from the National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases, from a joint award to M.J.M. and R.G.K. (R01DK127637). M.J.M. also acknowledges support from the National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases (R01DK113103), and the U.S. Department of Veterans Affairs Biomedical Laboratory Research and Development Service (I01BX005113).

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

1.
Merrins
MJ
,
Corkey
BE
,
Kibbey
RG
,
Prentki
M.
Metabolic cycles and signals for insulin secretion
.
Cell Metab
2022
;
34
:
947
968
2.
Rutter
GA
,
Sweet
IR
.
Glucose regulation of β-cell KATP channels: is a new model needed
?
Diabetes
2024
;
73
:
849
855
3.
Corradi
J
,
Thompson
B
,
Fletcher
PA
,
Bertram
R
,
Sherman
AS
,
Satin
LS.
KATP channel activity and slow oscillations in pancreatic beta cells are regulated by mitochondrial ATP production
.
J Physiol
2023
;
601
:
5655
5667
4.
Lang
AL
,
Nissanka
N
,
Louzada
RA
, et al
.
A defect in mitochondrial complex III but not in complexes I or IV causes early β-cell dysfunction and hyperglycemia in mice
.
Diabetes
2023
;
72
:
1262
1276
5.
Jung
SK
,
Kauri
LM
,
Qian
WJ
,
Kennedy
RT.
Correlated oscillations in glucose consumption, oxygen consumption, and intracellular free Ca(2+) in single islets of Langerhans
.
J Biol Chem
2000
;
275
:
6642
6650
6.
Kennedy
RT
,
Kauri
LM
,
Dahlgren
GM
,
Jung
SK.
Metabolic oscillations in beta-cells
.
Diabetes
2002
;
51
(
Suppl. 1
):
S152
S161
7.
Luciani
DS
,
Misler
S
,
Polonsky
KS.
Ca2+ controls slow NAD(P)H oscillations in glucose-stimulated mouse pancreatic islets
.
J Physiol
2006
;
572
:
379
392
8.
Matthews
DR
,
Naylor
BA
,
Jones
RG
,
Ward
GM
,
Turner
RC.
Pulsatile insulin has greater hypoglycemic effect than continuous delivery
.
Diabetes
1983
;
32
:
617
621
9.
Song
SH
,
McIntyre
SS
,
Shah
H
,
Veldhuis
JD
,
Hayes
PC
,
Butler
PC.
Direct measurement of pulsatile insulin secretion from the portal vein in human subjects
.
J Clin Endocrinol Metab
2000
;
85
:
4491
4499
10.
Krippeit-Drews
P
,
Düfer
M
,
Drews
G.
Parallel oscillations of intracellular calcium activity and mitochondrial membrane potential in mouse pancreatic B-cells
.
Biochem Biophys Res Commun
2000
;
267
:
179
183
11.
Lewandowski
SL
,
Cardone
RL
,
Foster
HR
, et al
.
Pyruvate kinase controls signal strength in the insulin secretory pathway
.
Cell Metab
2020
;
32
:
736
750.e5
12.
Merrins
MJ
,
Bertram
R
,
Sherman
A
,
Satin
LS.
Phosphofructo-2-kinase/fructose-2,6-bisphosphatase modulates oscillations of pancreatic islet metabolism
.
PLoS One
2012
;
7
:
e34036
13.
Merrins
MJ
,
Van Dyke
AR
,
Mapp
AK
,
Rizzo
MA
,
Satin
LS.
Direct measurements of oscillatory glycolysis in pancreatic islet β-cells using novel fluorescence resonance energy transfer (FRET) biosensors for pyruvate kinase M2 activity
.
J Biol Chem
2013
;
288
:
33312
33322
14.
Foster
HR
,
Ho
T
,
Potapenko
E
, et al
.
β-Cell deletion of the PKm1 and PKm2 isoforms of pyruvate kinase in mice reveals their essential role as nutrient sensors for the KATP channel
.
eLife
2022
;
11
:
e79422
15.
Abulizi
A
,
Cardone
RL
,
Stark
R
, et al
.
Multi-tissue acceleration of the mitochondrial phosphoenolpyruvate cycle improves whole-body metabolic health
.
Cell Metab
2020
;
32
:
751
766.e11
16.
Ho
T
,
Potapenko
E
,
Davis
DB
,
Merrins
MJ.
A plasma membrane-associated glycolytic metabolon is functionally coupled to KATP channels in pancreatic α and β cells from humans and mice
.
Cell Rep
2023
;
42
:
112394
17.
Weiss
JN
,
Lamp
ST.
Glycolysis preferentially inhibits ATP-sensitive K+ channels in isolated guinea pig cardiac myocytes
.
Science
1987
;
238
:
67
69
18.
Weiss
JN
,
Lamp
ST.
Cardiac ATP-sensitive K+ channels. Evidence for preferential regulation by glycolysis
.
J Gen Physiol
1989
;
94
:
911
935
19.
Dhar-Chowdhury
P
,
Malester
B
,
Rajacic
P
,
Coetzee
WA.
The regulation of ion channels and transporters by glycolytically derived ATP
.
Cell Mol Life Sci
2007
;
64
:
3069
3083
20.
Dhar-Chowdhury
P
,
Harrell
MD
,
Han
SY
, et al
.
The glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase, triose-phosphate isomerase, and pyruvate kinase are components of the K(ATP) channel macromolecular complex and regulate its function
.
J Biol Chem
2005
;
280
:
38464
38470
21.
Hong
M
,
Kefaloyianni
E
,
Bao
L
, et al
.
Cardiac ATP-sensitive K+ channel associates with the glycolytic enzyme complex
.
FASEB J
2011
;
25
:
2456
2467
22.
Polino
AJ
,
Sviben
S
,
Melena
I
,
Piston
DW
,
Hughes
JW.
Scanning electron microscopy of human islet cilia
.
Proc Natl Acad Sci U S A
2023
;
120
:
e2302624120
23.
Li
J
,
Shuai
HY
,
Gylfe
E
,
Tengholm
A.
Oscillations of sub-membrane ATP in glucose-stimulated beta cells depend on negative feedback from Ca(2+)
.
Diabetologia
2013
;
56
:
1577
1586
24.
Kennedy
HJ
,
Pouli
AE
,
Ainscow
EK
,
Jouaville
LS
,
Rizzuto
R
,
Rutter
GA.
Glucose generates sub-plasma membrane ATP microdomains in single islet beta-cells. Potential role for strategically located mitochondria
.
J Biol Chem
1999
;
274
:
13281
13291
25.
Niki
I
,
Ashcroft
FM
,
Ashcroft
SJ.
The dependence on intracellular ATP concentration of ATP-sensitive K-channels and of Na,K-ATPase in intact HIT-T15 beta-cells
.
FEBS Lett
1989
;
257
:
361
364
26.
Stanley
CA
,
Lieu
YK
,
Hsu
BY
, et al
.
Hyperinsulinism and hyperammonemia in infants with regulatory mutations of the glutamate dehydrogenase gene
.
N Engl J Med
1998
;
338
:
1352
1357
27.
Nicholls
DG.
The pancreatic β-cell: a bioenergetic perspective
.
Physiol Rev
2016
;
96
:
1385
1447
28.
Affourtit
C
,
Brand
MD.
Stronger control of ATP/ADP by proton leak in pancreatic beta-cells than skeletal muscle mitochondria
.
Biochem J
2006
;
393
:
151
159
29.
Stark
R
,
Kibbey
RG.
The mitochondrial isoform of phosphoenolpyruvate carboxykinase (PEPCK-M) and glucose homeostasis: has it been overlooked?
Biochim Biophys Acta
2014
;
1840
:
1313
1330
30.
Kibbey
RG
,
Pongratz
RL
,
Romanelli
AJ
,
Wollheim
CB
,
Cline
GW
,
Shulman
GI.
Mitochondrial GTP regulates glucose-stimulated insulin secretion
.
Cell Metab
2007
;
5
:
253
264
31.
Jesinkey
SR
,
Madiraju
AK
,
Alves
TC
, et al
.
Mitochondrial GTP links nutrient sensing to β cell health, mitochondrial morphology, and insulin secretion independent of OxPhos
.
Cell Rep
2019
;
28
:
759
772.e10
32.
Stark
R
,
Pasquel
F
,
Turcu
A
, et al
.
Phosphoenolpyruvate cycling via mitochondrial phosphoenolpyruvate carboxykinase links anaplerosis and mitochondrial GTP with insulin secretion
.
J Biol Chem
2009
;
284
:
26578
26590
33.
Taddeo
EP
,
Alsabeeh
N
,
Baghdasarian
S
, et al
.
Mitochondrial proton leak regulated by cyclophilin D elevates insulin secretion in islets at nonstimulatory glucose levels
.
Diabetes
2020
;
69
:
131
145
34.
Sweet
IR
,
Li
G
,
Najafi
H
,
Berner
D
,
Matschinsky
FM.
Effect of a glucokinase inhibitor on energy production and insulin release in pancreatic islets
.
Am J Physiol
1996
;
271
:
E606
E625
35.
Gregg
T
,
Poudel
C
,
Schmidt
BA
, et al
.
Pancreatic β-cells from mice offset age-associated mitochondrial deficiency with reduced KATP channel activity
.
Diabetes
2016
;
65
:
2700
2710
36.
Jung
SR
,
Kuok
ITD
,
Couron
D
, et al
.
Reduced cytochrome C is an essential regulator of sustained insulin secretion by pancreatic islets
.
J Biol Chem
2011
;
286
:
17422
17434
37.
Affourtit
C
,
Alberts
B
,
Barlow
J
,
Carré
JE
,
Wynne
AG.
Control of pancreatic β-cell bioenergetics
.
Biochem Soc Trans
2018
;
46
:
555
564
38.
Wikstrom
JD
,
Sereda
SB
,
Stiles
L
, et al
.
A novel high-throughput assay for islet respiration reveals uncoupling of rodent and human islets
.
PLoS One
2012
;
7
:
e33023
39.
Wescott
AP
,
Kao
JPY
,
Lederer
WJ
,
Boyman
L.
Voltage-energized calcium-sensitive ATP production by mitochondria
.
Nat Metab
2019
;
1
:
975
984
40.
Szibor
M
,
Gizatullina
Z
,
Gainutdinov
T
, et al
.
Cytosolic, but not matrix, calcium is essential for adjustment of mitochondrial pyruvate supply
.
J Biol Chem
2020
;
295
:
4383
4397
41.
Regeenes
R
,
Wang
Y
,
Piro
A
, et al
.
Design of an islet-on-a-chip device reveals glucose-stimulated respiration is substrate limited by glycolytic flux through PKM2
.
bioRxiv.
3 March
2022
[preprint]. DOI: 10.1101/2022.03.02.482671v1
42.
Behrman
EJ
,
Gopalan
V.
Phosphoenolpyruvate: an end to hand-waving
.
Biochem Mol Biol Educ
2008
;
36
:
323
324
43.
Pizarro-Delgado
J
,
Deeney
JT
,
Corkey
BE
,
Tamarit-Rodriguez
J.
Direct stimulation of islet insulin secretion by glycolytic and mitochondrial metabolites in KCl-depolarized islets
.
PLoS One
2016
;
11
:
e0166111
Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. More information is available at https://www.diabetesjournals.org/journals/pages/license.