Hallmarks of mature β cells are restricted proliferation and a highly energetic secretory state. Paradoxically, cyclin-dependent kinase 2 (CDK2) is synthesized throughout adulthood, its cytosolic localization raising the likelihood of cell cycle-independent functions. In the absence of any changes in β cell mass, maturity, or proliferation, genetic deletion of Cdk2 in adult β cells enhanced insulin secretion from isolated islets and improved glucose tolerance in vivo. At the single β cell level, CDK2 restricts insulin secretion by increasing KATP conductance, raising the set point for membrane depolarization in response to activation of the phosphoenolpyruvate (PEP) cycle with mitochondrial fuels. In parallel with reduced β cell recruitment, CDK2 restricts oxidative glucose metabolism while promoting glucose-dependent amplification of insulin secretion. This study provides evidence of essential, non-canonical functions of CDK2 in the secretory pathways of quiescent β cells.
Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Pyruvate kinase (PK) and the phosphoenolpyruvate (PEP) cycle play key roles in nutrient-stimulated KATP channel closure and insulin secretion. To identify the PK isoforms involved, we generated mice lacking β-cell PKm1, PKm2, and mitochondrial PEP carboxykinase (PCK2) that generates mitochondrial PEP. Glucose metabolism was found to generate both glycolytic and mitochondrially derived PEP, which triggers KATP closure through local PKm1 and PKm2 signaling at the plasma membrane. Amino acids, which generate mitochondrial PEP without producing glycolytic fructose 1,6-bisphosphate to allosterically activate PKm2, signal through PKm1 to raise ATP/ADP, close KATP channels, and stimulate insulin secretion. Raising cytosolic ATP/ADP with amino acids is insufficient to close KATP channels in the absence of PK activity or PCK2, indicating that KATP channels are primarily regulated by PEP that provides ATP via plasma membrane-associated PK, rather than mitochondrially derived ATP. Following membrane depolarization, the PEP cycle is involved in an 'off-switch' that facilitates KATP channel reopening and Ca2+ extrusion, as shown by PK activation experiments and β-cell PCK2 deletion, which prolongs Ca2+ oscillations and increases insulin secretion. In conclusion, the differential response of PKm1 and PKm2 to the glycolytic and mitochondrial sources of PEP influences the β-cell nutrient response, and controls the oscillatory cycle regulating insulin secretion. Editor's evaluation This manuscript employs in vitro studies and elegant mouse models to detail how specific pyruvate kinase isoforms impact pancreatic β-cell ATP/ADP levels, ATP-sensitive K+ channel (KATP channel) activity, calcium handling, and insulin secretion. This is an important study that challenges the current paradigms of KATP-channel regulation, the major signaling mechanism through which pancreatic β cells couple blood glucose levels to insulin release. Future studies will determine whether similar mechanisms are used in human pancreatic β cells. https://doi.org/10.7554/eLife.79422.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Maintenance of euglycemia relies on β-cells to couple nutrient sensing with appropriate insulin secretion. Insulin release is stimulated by the metabolism-dependent closure of ATP-sensitive K+ (KATP) channels (Ashcroft et al., 1984; Cook and Hales, 1984; Misler et al., 1986; Rorsman and Trube, 1985), which triggers Ca2+ influx and exocytosis (Anderson and Long, 1947; Grodsky et al., 1963). Contrary to what is often believed, the glucose-induced signaling process in β-cells has not been largely solved, and the entrenched model implicating a rise in mitochondrially derived ATP driving KATP channel closure (Campbell and Newgard, 2021; Prentki et al., 2013) is incomplete and possibly wrong, the main reason being that it does not consider other sources of local ATP production that may be key for signaling (Corkey, 2020; Lewandowski et al., 2020; Merrins et al., 2022). The recent discovery that pyruvate kinase (PK), which converts ADP and phosphoenolpyruvate (PEP) to ATP and pyruvate, is present on the β-cell plasma membrane where it is sufficient to raise sub-plasma membrane ATP/ADP (ATP/ADPpm) and close KATP channels (Lewandowski et al., 2020) provides an alternative mechanism to oxidative phosphorylation for KATP channel regulation. Based on this finding, Lewandowski et al., 2020, proposed a revised model of β-cell fuel sensing, which we refer to here as the MitoCat-MitoOx model, that is relevant to both rodent and human islets and the in vivo context (Abulizi et al., 2020; Lewandowski et al., 2020; Merrins et al., 2022). In the MitoCat-MitoOx model of β-cell metabolic signaling, Ca2+ and ADP availability dictate the metabolic cycles that preferentially occur during the triggering or secretory phases of glucose-stimulated oscillations (Figure 1—figure supplement 1). The triggering phase, referred to as MitoCat (a.k.a. MitoSynth; Lewandowski et al., 2020), is named for the matched processes of anaplerosis (i.e., the net filling of TCA cycle intermediates) and cataplerosis (i.e., the egress of TCA cycle intermediates to the cytosol). During this electrically silent phase of metabolism, the favorable bioenergetics of PEP metabolism (ΔG°=–14.8 kcal/mol for PEP vs. –7.3 for ATP) by PK progressively increases the ATP/ADP ratio, and by lowering ADP slows oxidative phosphorylation. The shift to a higher mitochondrial membrane potential (ΔΨm) elevates the NADH/NAD+ ratio in the mitochondrial matrix and slows the TCA cycle, increasing acetyl-CoA that allosterically activates pyruvate carboxylase, the anaplerotic consumer of pyruvate that fuels oxaloacetate-dependent PEP synthesis by mitochondrial PEP carboxykinase (PCK2). The return of mitochondrial PEP to the cytosol completes the 'PEP cycle' that helps fuel PK, which raises ATP/ADPpm to close KATP channels. Following membrane depolarization and Ca2+ influx, the increased workload (ATP hydrolysis) associated with ion pumping and exocytosis elevates cytosolic ADP, which activates oxidative phosphorylation to produce ATP that sustains insulin secretion in a phase referred to as MitoOx. An unresolved aspect of this model is whether plasma membrane-compartmentalized PK activity is required to close KATP channels. This question is important because in the current canonical model of fuel-induced insulin secretion, an increase in the bulk cytosolic ATP/ADP ratio (ATP/ADPc) is generally assumed to close KATP channels. In the MitoCat-MitoOx model, PK has two possible sources of PEP that may differentially regulate KATP closure: glycolytic PEP produced by enolase, and mitochondrial PEP produced by PCK2 in response to anaplerosis (Figure 1A). About 40% of glucose-derived PEP is generated by PCK2 in the PEP cycle and is closely linked to insulin secretion (Abulizi et al., 2020; Jesinkey et al., 2019; Stark et al., 2009). However, it remains unclear how the PEP cycle influences glucose-stimulated oscillations. We hypothesize that mitochondrial PEP derived from PCK2 may provide a glycolysis-independent mechanism by which PK rapidly increases ATP/ADPpm locally at the KATP channel in response to amino acids, which are potent anaplerotic fuels. Figure 1 with 3 supplements see all Download asset Open asset Generation of mouse models to probe the functions of PKm1, PKm2, and phosphoenolpyruvate carboxykinase (PCK2) in β-cells. (A) Hypothesized model in which pyruvate kinase (PK) in the KATP microcompartment is fueled by two sources of phosphoenolpyruvate (PEP) – glycolytic PEP generated by enolase, and mitochondrial PEP generated by PCK2 in response to anaplerotic fuels. β-Cells express three isoforms of PK, constitutively active PKm1, and allosterically recruitable PKm2 and PKL that are activated by endogenous fructose 1,6-bisphosphate (FBP) or pharmacologic PK activators (PKa). (B–D) Quantification of knockdown efficiency in islet lysates from PKm1-βKO (B), PKm2-βKO (C), and Pck2-βKO mice (D) (n=4 mice for PKm1- and PCK2-βKO, n=6 mice for PKm2-βKO). (E–G) Intraperitoneal glucose tolerance tests (GTT, 1 g/kg) of PKm1-βKO mice (n=9) and littermate controls (n=8) (E), PKm2-βKO mice (n=7) and littermate controls (n=7) (F), and Pck2-βKO mice (n=10) and littermate controls (n=7) (G) following an overnight fast. (H–I) PK activity in islet lysates of PKm1-βKO (H) and PKm2-βKO mice (I) in response to FBP (80 µM) and PKa (10 µM TEPP-46) (n=2 replicates from 6 mice/group). See Figure 1—source data 1 and Figure 1—source data 2 for source data. Data are shown as mean ± SEM. #p<0.01, *p<0.05, **p<0.01, ***p<0.001, **** p<0.0001 by t-test (B–G) or two-way ANOVA (H–I). Figure 1—source data 1 Western blot source data. https://cdn.elifesciences.org/articles/79422/elife-79422-fig1-data1-v2.zip Download elife-79422-fig1-data1-v2.zip Figure 1—source data 2 Source data for Western blots, glucose tolerance tests, and PK activity assays associated with Figure 1. https://cdn.elifesciences.org/articles/79422/elife-79422-fig1-data2-v2.xlsx Download elife-79422-fig1-data2-v2.xlsx The isoforms of PK, each with different activities and mechanisms of control, may differentially regulate KATP channels (Figure 1A). β-Cells express the constitutively active PKm1 as well as two allosterically recruitable isoforms, PKm2 and PKL, which are activated by glycolytic fructose-1,6-bisphosphate (FBP) generated upstream by the phosphofructokinase reaction (DiGruccio et al., 2016; MacDonald and Chang, 1985; Mitok et al., 2018). Pharmacologic PK activators (PKa), which lower the Km of PKm2 for PEP and increase the Vmax (Anastasiou et al., 2012), increase the frequency of glucose-stimulated Ca2+ and ATP/ADP oscillations and potentiate nutrient-stimulated insulin secretion from rodent and human islets (Abulizi et al., 2020; Lewandowski et al., 2020). Much less is known about the PKm1 isoform, which due to its constitutive (FBP-insensitive) activity might be ideal in situations of high oxidative workload, as in cardiac myocytes (Li et al., 2021). β-Cells may shift their reliance upon different PK isoforms throughout the oscillatory cycle, as the levels of glycolytic FBP rise during MitoCat and fall during MitoOx (Lewandowski et al., 2020; Merrins et al., 2016; Merrins et al., 2013). Here, we show that PK is essential for KATP closure – amino acids that effectively raise ATP/ADPc cannot close KATP channels without PK. We further demonstrate that both PKm1 and PKm2 are active in the KATP channel microcompartment with at least two required functions. First, spatial privilege provides redundancy in the β-cell glucose response, by permitting the minor PKm2 isoform, when activated by FBP, to transmit the signal from glucose to KATP despite contributing only a small fraction of the whole cell PK activity. Second, the composition of PK isoforms within the KATP compartment tunes the β-cell response to amino acids, which provide mitochondrial PEP for PKm1 without also generating the FBP needed to allosterically activate PKm2. Using β-cell PCK2 deletion, we found that mitochondrially derived PEP signals to the plasma membrane PK-KATP microcompartment during MitoCat, and facilitates Ca2+ extrusion during MitoOx. These studies support the MitoCat-MitoOx model of oscillatory metabolism, and identify unique functions of the PKm1- and PKm2-driven PEP cycles in β-cell nutrient signaling. Results β-Cell PKm1 accounts for >90% of total PK activity in mouse islets, with <10% from β-cell PKm2 and no discernable contribution from PKL We generated β-cell-specific PKm1 and PKm2 knockout mice by breeding Ins1Cre mice (Thorens et al., 2015) with Pkm1f/f mice (Davidson et al., 2021; Li et al., 2021) (PKm1-βKO) or Pkm2f/f mice (Israelsen et al., 2013) (PKm2-βKO). PKm1 protein was reduced by 90% in PKm1-βKO islets, and PKm2 was not significantly increased compared to littermate Ins1Cre controls (Figure 1B). Expression of PKm2 protein fell by 94% in PKm2-βKO islets, while PKm1 increased by 29% (Figure 1C). This partial compensation is expected since PKm1 and PKm2 are alternative splice variants of the Pkm gene (Li et al., 2021; Israelsen et al., 2013). We generated Pck2f/f mice (Figure 1—figure supplement 2) and crossed them with Ucn3 Cre mice to facilitate postnatal β-cell deletion without the need for tamoxifen (Adams et al., 2021; van der Meulen et al., 2017). Islet PCK2 protein dropped by 99% in the Pck2-βKO compared to Pck2f/f littermate controls (Figure 1D). None of these knockout mice were glucose intolerant (Figure 1E–G), nor did they exhibit changes in meal tolerance by oral gavage (Figure 1—figure supplement 3A-C). The contributions of each PK isoform relative to total PK activity was determined in the islet lysates. The endogenous allosteric metabolite, FBP, and pharmacologic PKa such as TEPP-46 Abulizi et al., 2020; Anastasiou et al., 2012; Lewandowski et al., 2020 have no impact on PKm1 but substantially lower the Km and raise Vmax of PKm2 or PKL (Lewandowski et al., 2020). Control islet lysates had a Km for PEP of 140±14 µM that was reduced in the presence of exogenous FBP (Km, 100 μM±10 µM) or PKa (Km, 90±13 µM), while the Vmax increased by about one-third (control, 1.05±0.019 µmol/min; FBP, 1.28±0.006 µmol/min; PKa, 1.36±0.006 µmol/min). The Vmax for islet PK activity from PKm1-βKO mice decreased by 97% compared to controls (Figure 1H), and was too low to estimate Km accurately. The residual PK remained sensitive to activation by both FBP and PKa, identifying an allosterically recruitable PK pool that accounts for only about 10% of the PK activity present in control islets (Figure 1H). Conversely, β-cell PKm2 deletion lowered islet lysate PK Vmax by ~20% in the absence of activators, and eliminated both the Km and Vmax response to PKa and FBP (Figure 1I), thus ruling out any measurable PKL activity. Taken together, mouse islet PK activity is composed of >90% PKm1, with a variable contribution from PKm2 depending on the FBP level. If only considered in terms of total cellular activity related to nutrient-induced insulin secretion (i.e. in the absence of any compartmentalized functions), PKm1 should be dominant over PKm2 under all physiologic conditions. The fact that PKm1-βKO mice maintain metabolic health with unaltered glucose tolerance into adulthood suggests that the remaining PK activity is sufficient for β-cell function, and led us to hypothesize that both PKm1 and PKm2 function in the KATP channel microcompartment. Both PKm1 and PKm2 are associated with the plasma membrane and locally direct KATP channel closure, however PKm2 requires allosteric activation even at high PEP levels We previously demonstrated that PEP, in the presence of saturating ADP concentrations, can close KATP channels in mouse and human β-cells (Lewandowski et al., 2020). This suggests that PK is present near KATP and locally lowers ADP and raises ATP to close KATP channels. Excised patch-clamp experiments, which expose the inside of the plasma membrane to the bath solution (i.e. the inside-out mode), provide both the location of endogenous PK and its functional coupling to KATP channels in native β-cell membranes. This approach was applied in combination with β-cell deletion of PKm1 and PKm2 to directly identify the isoforms of the enzyme present in the KATP microdomain. KATP channels were identified by inhibition with 1 mM ATP, which blocked the spontaneous opening that occurs after patch excision (Figure 2A). Channel activity was restored using a test solution containing 0.5 mM ADP and 0.1 mM ATP. In control β-cells, the further addition of 5 mM PEP closed KATP, as shown by a 77% reduction in the total power (a term reflecting both the frequency and channel open time) (Figure 2A), compared with only a 29% reduction in PKm1-βKO cells (Figure 2B). Note that KATP channel closure occurred in control β-cells despite the continuous deluge of the channel-opener MgADP. Thus, it is not the PEP itself, but the PK activity in the KATP microcompartment that is responsible for KATP closure. Figure 2 Download asset Open asset Plasma membrane KATP channels are locally regulated by a combination of PKm1 and allosterically activated PKm2. (A–E) KATP channel activity (holding potential = –50 mV) quantified in terms of power, frequency, and open time. Applying the substrates for pyruvate kinase (PK) closes KATP channels in excised patches of β-cell plasma membrane from control mice (n=14 recordings from 4 mice) (A). Defective KATP channel closure in β-cells from PKm1-KO (n=20 recordings from 5 mice) (B) is rescued by PK activator (PKa) pretreatment (n=6 recordings from 3 mice) (C) and acute PKa application (n=7 recordings from 3 mice) (D). PKm1 is sufficient for KATP closure in β-cells from PKm2-βKO (n=6 recordings from 3 mice) (E). ATP, 1 mM; ADP, 0.5 mM ADP+ 0.1 mM ATP; PEP, 5 mM; PKa, 10 µM TEPP-46. See Figure 2—source data 1 for source data. Data are shown as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by paired t-test. (*)p<0.05 by unpaired t-test in (H). Figure 2—source data 1 Source data for excised patch clamp experiments in Figure 2. https://cdn.elifesciences.org/articles/79422/elife-79422-fig2-data1-v2.xlsx Download elife-79422-fig2-data1-v2.xlsx To test for a role of PKm2 in the KATP microcompartment, PKm1-βKO cells were preincubated in the presence of 10 μM PKa, which restored PEP-dependent KATP channel closure to the same extent as the control (1 mM ATP) (Figure 2C). PKa had a similar effect when applied acutely (Figure 2D), indicating that PKm2 does not require allosteric activation to localize to the plasma membrane. Although the PEP concentration is estimated to be 1 mM in rat islets (Sugden and Ashcroft, 1977), 5 mM PEP was chosen to exceed the Km of PKm2 in the absence of FBP (Lewandowski et al., 2020). Therefore, the response of PKm2 to PKa at high PEP levels indicates that KATP closure requires an additional increase in the Vmax via either allosteric activation of individual subunits of PKm2, or perhaps more likely, that the functional interaction is sensitive to the quaternary structure of PKm2. β-Cells lacking PKm2 maintained channel closure with the same power as 1 mM ATP, which is attributable to the sufficiency of endogenous PKm1 (Figure 2E). Thus, metabolic compartmentation of PKm1 and PKm2 to the plasma membrane provides a redundant mechanism of KATP channel regulation when PKm2 is allosterically activated, as well as a compelling explanation for the ability of PKm1-βKO mice to tolerate a near-complete loss of β-cell PK activity (Figure 1E and H). PKm1 and PKm2 are redundant for glucose-dependent Ca2+ influx The rescue of PKm1 deficiency by PKa in the KATP microcompartment (Figure 2C–D) suggests that PKm1 and PKm2 exert shared control over KATP closure, provided that glucose is present to generate FBP to activate PKm2. To test this further we examined Ca2+ dynamics with FuraRed while using a near-infrared dye, DiR, to facilitate simultaneous imaging of PKm1-, PKm2-, and PCK2-βKO islets with their littermate controls (Figure 3A). β-Cell deletion of PKm1 revealed no difference in the oscillatory period or amplitude and a modest increase in the fraction of each oscillation spent in the electrically active state (i.e. the duty cycle) (Figure 3B). β-Cell PKm2 deletion increased the period of Ca2+ oscillations, while having no impact on the amplitude or the duty cycle (Figure 3D). In addition, PKm1 and PKm2 knockouts had no discernable difference in first-phase Ca2+ parameters (i.e. time to depolarization, amplitude, and duration of first phase) following an acute rise in glucose from 2 to 10 mM (Figure 3C and E). These data confirm in situ that PKm1 and PKm2 are largely redundant at high glucose. Figure 3 with 1 supplement see all Download asset Open asset PKm2 and phosphoenolpyruvate carboxykinase (PCK2), but not PKm1, have metabolic control over first-phase and steady-state Ca2+ influx in response to glucose. (A) Barcoding of islet preparations with near-IR dye (DiR) permits simultaneous timelapse imaging of islet Ca2+ dynamics of control and βKO mice (scale bar = 200 µm) (left). A representative trace illustrates the cataplerotic triggering phase (MitoCat) and oxidative secretory phases (MitoOx) of steady-state Ca2+ oscillations in the presence of 10 mM glucose and 1 mM leucine (right). Gray line denotes the period. (B, D, F, H) Representative traces and quantification of period, amplitude, and duty cycle of steady-state Ca2+ oscillations in islets from PKm1-βKO (n=94 islets from 3 mice) and littermate controls (n=91 islets from 3 mice) (B), PKm2-βKO (n=118 islets from 4 mice) and littermate controls (n=111 islets from 4 mice) (D), control mice treated with PK activator (PKa) (10 μM TEPP-46) (n=88 islets from 3 mice) (F), and PCK2-βKO (n=74 islets from 3 mice) and littermate controls (n=77 islets from 3 mice) (H). The bath solution (PSS) contained 10 mM glucose (10G) and 1 mM leucine. Scale bars: 0.1 FuraRed excitation ratio (R430/500). (C, E, G, I) Representative Ca2+ traces and quantification of time to depolarization (a), first-phase amplitude (b), and first-phase duration (c) in islets from PKm1-βKO (n=144 islets from 6 mice) and littermate controls (n=150 islets from 6 mice) (C), PKm2-βKO (n=52 islets from 2 mice) and littermate controls (n=55 islets from 2 mice) (E), PKa-treated (10 μM TEPP-46) (n=161 islets from 9 mice) and vehicle controls (n=212 islets from 9 mice) (G), and PCK2-βKO (n=73 islets from 3 mice) and littermate controls (n=78 islets from 3 mice) (I). The bath solution (PSS) contained 1 mM leucine and 2.7 mM (2.7G) and 10 mM glucose (10G) as indicated. Scale bars: 0.1 FuraRed excitation ratio (R430/500). See Figure 3—source data 1 for source data. Data are shown as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by unpaired t-test (A–C, and E–I) and paired t-test (D). Figure 3—source data 1 Source data for calcium imaging assays in Figure 3. https://cdn.elifesciences.org/articles/79422/elife-79422-fig3-data1-v2.xlsx Download elife-79422-fig3-data1-v2.xlsx PK and the PEP cycle are implicated in both on- and off-switches for Ca2+ influx To study the interaction of PK with the PEP cycle, we performed islet Ca2+ measurements using PKa and PCK2-βKO islets, in the latter case using islet barcoding to simultaneously image islets isolated from littermate controls. Consistent with the ability of allosteric PKm2 activation to accelerate KATP closure, acute application of PKa to wild-type islets reduced the period as well as the amplitude of the steady-state glucose-induced Ca2+ oscillations (Figure 3F and Figure 3—figure supplement 1A). However, we noticed that PKa shortened the time spent in MitoCat and to a greater degree, MitoOx (Figure 3—figure supplement 1B), leading to a modest reduction in the duty cycle as well as a more significant reduction in the period of the oscillation (Figure 3F). These observations suggest that the PKm2-driven PEP cycle regulates the onset, and even more strongly, the termination of Ca2+ influx. Consistently, in PCK2-βKO islets where mitochondrial PEP production is inhibited, both the period and amplitude of glucose-stimulated Ca2+ oscillations were increased relative to controls islets (Figure 3H). Although the duty cycle also increased, it was only by a small margin. The period lengthening occurred in part from an increased duration of MitoCat, and especially from an increased duration of MitoOx (Figure 3—figure supplement 1C). Taken together, these data indicate that PKm2 controls both an 'on-switch' and an 'off-switch' for Ca2+ oscillations, both of which depend on the mitochondrial production of PEP. While above experiments examined conditions at a fixed elevated glucose concentration (10 mM), we also investigated Ca2+ dynamics following the transition from low to high glucose where first-phase insulin secretion is observed. Preincubation of control islets with PKa reduced the time to depolarization as well as the duration of the first-phase Ca2+ influx (Figure 3G). Conversely, depolarization was delayed in PCK2-βKO islets (Figure 3I). In this case, the duration of the first-phase Ca2+ pulse was not calculated since nearly 60% of PCK2-βKO islets failed to exit the first-phase plateau in order to begin oscillations, as compared with only 27% of control islets (Figure 3I). In other words, while the PCK2 knockout had a weaker first-phase Ca2+ rise, it had a much longer plateau that failed to turn off effectively. Hence, PKm2 activation and PCK2 serve as on-switches for promoting glucose-stimulated Ca2+ influx during the triggering phase (MitoCat), and with a quantitatively larger effect, off-switches during the secretory phase (MitoOx). Mitochondrial PCK2 is essential for amino acids to promote a rise in cytosolic ATP/ADP To determine whether PCK2, PKm1, or PKm2 are essential for the rise in the cytosolic ATP/ADP ratio generated by high glucose or amino acids, we used β-cell-specific expression of Perceval-HR biosensors to measure ATP/ADPc. We found that, as with Ca2+, there were no significant differences in glucose-stimulated ATP/ADPc detected in the PKm1- or PKm2-βKO islets (Figure 4A and B), demonstrating the redundancy of the two isoforms at high glucose for ATP/ADPc generation. Similarly, we detected no significant difference in glucose-stimulated ATP/ADPc in islets from the PCK2-βKO (Figure 4C). Figure 4 Download asset Open asset Restriction of the glycolytic phosphoenolpyruvate (PEP) supply reveals the importance of PEP carboxykinase (PCK2) for cytosolic ATP/ADP. Average β-cell ATP/ADPc in islets from PCK2-βKO (A, D, G), PKm1-βKO (B, F, H), and PKm2-βKO (C, F, I) mice in response to glucose in the presence of 1 mM leucine (A–C) or mixed amino acids (AA) provided at three times their physiological concentrations (×1=Q, 0.6 mM; L, 0.5 mM; R, 0.2 mM; A, 2.1 mM) in the presence of 2.7 mM glucose (2.7G) to remove the enolase contribution to cytosolic PEP (D–I). Pyruvate kinase activator (PKa) (10 μM TEPP-46) was present in G, H, and I. ATP/ADPc is quantified as area under the curve from PCK2-βKO (A, n=65 islets from 3 mice; D, n=73 islets from 3 mice; G, n=71 islets from 3 mice) and littermate controls (A, n=63 islets from 3 mice; D, n=90 islets from 3 mice; G, n=77 islets from 3 mice); PKm1-βKO (B, n=88 islets from 3 mice; E, n=66 islets from 3 mice; H, n=72 islets from 3 mice) and littermate controls (B, n=92 islets from 3 mice; E, n=69 islets from 6 mice; H, n=69 islets from 3 mice); and PKm2-βKO (C, n=86 islets from 3 mice; F, n=99 islets from 3 mice; I, n=89 islets from 3 mice) and littermate controls (C, n=90 islets from 3 mice; F, n=100 islets from 6 mice; I, n=85 islets from 3 mice). Scale bars: 0.025 Perceval-HR excitation ratio for A–C and 0.1 Perceval-HR excitation ratio for D–I (R500/430). See Figure 4—source data 1 for source data. Data are shown as mean ± SEM. *p<0.05, ****p<0.0001 by t-test. Figure 4—source data 1 Source data for beta cell ATP/ADP measurements in Figure 4. https://cdn.elifesciences.org/articles/79422/elife-79422-fig4-data1-v2.xlsx Download elife-79422-fig4-data1-v2.xlsx Amino acids (AA) are obligate mitochondrial fuels that simultaneously feed oxidative and anaplerotic pathways. AA can be used as a tool for separating mechanistic components of the secretion mechanism because at low glucose they can, independently of glycolysis, raise ATP/ADPc and elicit KATP channel closure, Ca2+ influx, and insulin release. In particular, glutamine and leucine generate PEP via glutamate dehydrogenase (GDH)-mediated anaplerosis that is followed by PCK2-mediated cataplerosis of PEP (Kibbey et al., 2014; Stark et al., 2009). We first examined whether restriction of mitochondrial PEP production in PCK2-βKO islets impacts the cytosolic ATP/ADPc ratio. To limit glycolytic PEP, islets were incubated at 2.7 mM glucose. The islets were then stimulated with a mixture of AA including leucine and glutamine to allosterically activate and fuel GDH, respectively. Consistent with defective PEP cataplerosis, the ATP/ADPc response of PCK2-βKO islets was only 44% of control islets in response to AA (Figure 4D). In this setting of PCK2 depletion, pharmacologic PK activation did not recover any of the AA-induced ATP/ADPc response due to the absence of either a glycolytic or mitochondrial PEP source (Figure 4G). Comparatively, deletion of either PKm1 or PKm2 had only modest effects on the β-cell ATP/ADPc response to AA (Figure 4E and F). However, PKa completely recovered the AA-induced rise in ATP/ADPc in PKm1-βKO islets, in which the allosteric PKm2 isoform remains (Figure 4H). As expected, PK activation had no effect in PKm2-βKO islets (Figure 4I), confirming an on-target effect of TEPP-46. Mitochondrial fuels that stimulate a bulk rise in ATP/ADPc fail to close KATP in the absence of PK Mitochondria are located throughout the β-cell, including near the plasma membrane, where submembrane ATP microdomains have been observed (Griesche et al., 2019; Kennedy et al., 1999). Since PK is localized to the plasma membrane, we wondered whether during AA stimulation mitochondria can provide PEP to facilitate PK-dependent KATP closure, or alternatively, whether mitochondria can serve as a direct source of ATP for KATP channel closure. To determine whether mitochondrial PEP impacts the KATP channel microcompartment, we monitored KATP channel currents in intact β-cells in the cell-attached configuration in response to bath-applied AA at 2.7 mM glucose (Figure 5A). Mixed AA with or without PKa reduced KATP channel power (reflecting the total number of transported K+ ions) by ~75% in control β-cells (Figure 5B). However, no KATP closure was observed in the absence of PCK2, even with PKa present (Figure 5C). These findings indicate that mitochondrially derived PEP can signal to the KATP channel microcompartment, and is essential for KATP closure in response to AA. Figure 5 Download asset Open asset Mitochondrial phosphoenolpyruvate (PEP) signals to pyruvate kinase (PK) within the plasma membrane KATP channel microcompartment in intact β-cells. (A) Diagram of on-cell patch clamp method in intact β-cells with bath application of amino acids (AA). (B–F) Representative example traces and quantification of KATP channel closure in terms of normalized power, frequency, and open time for β-cells from control (n=10 recordings from 3 mice) (B), PCK2-βKO mice in the
Pyruvate kinase (PK) and the phosphoenolpyruvate (PEP) cycle play key roles in nutrient-stimulated KATP channel closure and insulin secretion. To identify the PK isoforms involved, we generated mice lacking β-cell PKm1, PKm2, and mitochondrial PEP carboxykinase (PCK2) that generates mitochondrial PEP. Glucose metabolism was found to generate both glycolytic and mitochondrially derived PEP, which triggers KATP closure through local PKm1 and PKm2 signaling at the plasma membrane. Amino acids, which generate mitochondrial PEP without producing glycolytic fructose 1,6-bisphosphate to allosterically activate PKm2, signal through PKm1 to raise ATP/ADP, close KATP channels, and stimulate insulin secretion. Raising cytosolic ATP/ADP with amino acids is insufficient to close KATP channels in the absence of PK activity or PCK2, indicating that KATP channels are primarily regulated by PEP that provides ATP via plasma membrane-associated PK, rather than mitochondrially derived ATP. Following membrane depolarization, the PEP cycle is involved in an 'off-switch' that facilitates KATP channel reopening and Ca2+ extrusion, as shown by PK activation experiments and β-cell PCK2 deletion, which prolongs Ca2+ oscillations and increases insulin secretion. In conclusion, the differential response of PKm1 and PKm2 to the glycolytic and mitochondrial sources of PEP influences the β-cell nutrient response, and controls the oscillatory cycle regulating insulin secretion.
Male mice lacking the androgen receptor (AR) in pancreatic β cells exhibit blunted glucose-stimulated insulin secretion (GSIS), leading to hyperglycemia. Testosterone activates an extranuclear AR in β cells to amplify glucagon-like peptide-1 (GLP-1) insulinotropic action. Here, we examined the architecture of AR targets that regulate GLP-1 insulinotropic action in male β cells. Testosterone cooperates with GLP-1 to enhance cAMP production at the plasma membrane and endosomes via: (1) increased mitochondrial production of CO2, activating the HCO3−-sensitive soluble adenylate cyclase; and (2) increased Gαs recruitment to GLP-1 receptor and AR complexes, activating transmembrane adenylate cyclase. Additionally, testosterone enhances GSIS in human islets via a focal adhesion kinase/SRC/phosphatidylinositol 3-kinase/mammalian target of rapamycin complex 2 actin remodeling cascade. We describe the testosterone-stimulated AR interactome, transcriptome, proteome, and metabolome that contribute to these effects. This study identifies AR genomic and non-genomic actions that enhance GLP-1-stimulated insulin exocytosis in male β cells.
The ATP-sensitive potassium channel (KATP) is a key regulator of membrane potential and islet hormone secretion. In ß-cells, it has been previously showed that the local activity of pyruvate kinase (PK) can sufficiently raise ATP/ADP to induce KATP closure, thus providing a another route to trigger membrane depolarization parallel to mitochondrial oxidative phosphorylation. Here, using direct single-channel measurements of KATP in both ß-and the lesser studied α-cells, we provide evidence for a larger network of metabolic enzymes that operate in the vicinity of KATP channels to fine-tune their activity. We found that the phosphofructokinase product fructose-1,6-bisphosphate (FBP), which oscillates in ß-cells, can induce KATP closure by its metabolism through the glycolytic chain including aldolase, GAPDH, phosphoglycerate kinase, phosphoglycerate mutase, enolase and finally PK. Strikingly, while withholding the substrates at GAPDH, we observed that FBP can independently inhibit KATP channels by direct action. In addition, we further provide evidence that creatine kinase (CK) activity can either oppose or enhance KATP closure, depending on the reaction direction. Finally, we demonstrated that lactate has an activating effect on KATP. In summary, these data provide evidence for the direct regulation of KATP by two metabolites, FBP and lactate, and identify two local ATP/ADP controllers, the glycolytic complex and the creatine kinase system in both ß-cells and α-cells. Disclosure T. Ho: None. E. Potapenko: None. M. J. Merrins: None.
The ATP-sensitive K+ channel (KATP) is a key regulator of hormone secretion from pancreatic islet endocrine cells. Using direct measurements of KATP channel activity in pancreatic β cells and the lesser-studied α cells, from both humans and mice, we demonstrate that a glycolytic metabolon locally controls KATP channels on the plasma membrane. The two ATP-consuming enzymes of upper glycolysis, glucokinase and phosphofructokinase, generate ADP that activates KATP. Substrate channeling of fructose 1,6-bisphosphate through the enzymes of lower glycolysis fuels pyruvate kinase, which directly consumes the ADP made by phosphofructokinase to raise ATP/ADP and close the channel. We further demonstrate the presence of a plasma membrane NAD+/NADH cycle, whereby lactate dehydrogenase is functionally coupled to glyceraldehyde-3-phosphate dehydrogenase. These studies provide direct electrophysiological evidence of a KATP-controlling glycolytic signaling complex and demonstrate its relevance to islet glucose sensing and excitability.Funding Information: This work was supported in part by the United States Department of Veterans Affairs Biomedical Laboratory Research and Development Service (I01B005113). Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: All procedures involving animals were approved by the Institutional Animal Care and Use Committees of the University of Wisconsin-Madison and the William S. Middleton Memorial Veterans Hospital, and followed the NIH Guide for the Care and Use of Laboratory Animals (8th ed. The National Academies Press. 2011.).
Pancreatic β-cells couple nutrient metabolism with appropriate insulin secretion. Here, we show that pyruvate kinase (PK), which converts ADP and phosphoenolpyruvate (PEP) into ATP and pyruvate, underlies β-cell sensing of both glycolytic and mitochondrial fuels. PK present at the plasma membrane is sufficient to close KATP channels and initiate calcium influx. Small-molecule PK activators increase β-cell oscillation frequency and potently amplify insulin secretion. By cyclically depriving mitochondria of ADP, PK restricts oxidative phosphorylation in favor of the mitochondrial PEP cycle with no net impact on glucose oxidation. Our findings support a compartmentalized model of β-cell metabolism in which PK locally generates the ATP/ADP threshold required for insulin secretion, and identify a potential therapeutic route for diabetes based on PK activation that would not be predicted by the β-cell consensus model.
Summary The ATP-sensitive K + channel (K ATP ) is a key regulator of hormone secretion from pancreatic islet endocrine cells. Using direct measurements of K ATP channel activity in pancreatic β cells and the lesser-studied α cells, from both humans and mice, we demonstrate that a glycolytic metabolon locally controls K ATP channels on the plasma membrane. The two ATP-consuming enzymes of upper glycolysis, glucokinase and phosphofructokinase, generate ADP that activates K ATP . Substrate channeling of fructose 1,6-bisphosphate through the enzymes of lower glycolysis fuels pyruvate kinase, which directly consumes the ADP made by phosphofructokinase to raise ATP/ADP and close the channel. We further demonstrate the presence of a plasma membrane NAD + /NADH cycle, whereby lactate dehydrogenase is functionally coupled to glyceraldehyde-3-phosphate dehydrogenase. These studies provide direct electrophysiological evidence of a K ATP -controlling glycolytic signaling complex and demonstrate its relevance to islet glucose sensing and excitability. Graphical abstract Ho et al . demonstrate that the enzymes of glycolysis, as well as lactate dehydrogenase, form a plasma membrane-associated metabolon with intrinsic ATP/ADP and NAD + /NADH cycles. The subcellular location of this signaling complex allows both ATP-consuming and ATP-producing enzymes to locally control the activity of ATP-sensitive K + channels in pancreatic α and β cells from humans and mice. Highlights K ATP channels are regulated by a glycolytic metabolon on the plasma membrane. Substrate channeling occurs between the consecutive enzymes of glycolysis. Upper glycolysis produces ADP that is used directly by lower glycolysis to make ATP. LDH and GADPH facilitate a plasma membrane-associated NAD + /NADH redox cycle.
Resurgence is defined as the recurrence of a previously reinforced and then extinguished target response when reducing or eliminating a more recently reinforced alternative response. In experiments with children and pigeons, we evaluated patterns of resurgence across and within sessions through decreases in reinforcer availability by challenging alternative responding with extinction and progressive‐ratio schedules. In Phase 1, we reinforced only target responding. In Phase 2, we extinguished target responding while reinforcing an alternative response. Finally, Phase 3 assessed resurgence by (a) extinguishing alternative responding versus (b) introducing a progressive‐ratio schedule of reinforcement for alternative responding. In both children and pigeons, resurgence of target responding occurred in both conditions but generally was greater when assessed during extinction than with progressive ratios. Importantly, within‐session patterns of resurgence did not differ between testing with progressive ratios and extinction. Resurgence with progressive ratios tended to be greater with longer durations between reinforcers but we observed similar findings with only simulated reinforcers during extinction testing. Therefore, the present investigation reveals that the events contributing to instances of resurgence remain to be understood, and presents an approach from which to examine variables influencing within‐session patterns of resurgence.
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