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SUMOylation reduces oxidative stress and preserves islet mass at the expense of robust insulin secretion. To investigate a role for the deSUMOylating enzyme sentrin-specific protease 1 (SENP1) following metabolic stress, we put pancreas/gut-specific SENP1 knockout (pSENP1-KO) mice on a high-fat diet (HFD). Male pSENP1-KO mice were more glucose intolerant following HFD than littermate controls but only in response to oral glucose. A similar phenotype was observed in females. Plasma glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1) responses were identical in pSENP1-KO and wild-type littermates, including the HFD-induced upregulation of GIP responses. Islet mass was not different, but insulin secretion and β-cell exocytotic responses to the GLP-1 receptor agonist exendin-4 (Ex4) and GIP were impaired in islets lacking SENP1. Glucagon secretion from pSENP1-KO islets was also reduced, so we generated β-cell–specific SENP1 KO mice. These phenocopied the pSENP1-KO mice with selective impairment in oral glucose tolerance following HFD, preserved islet mass expansion, and impaired β-cell exocytosis and insulin secretion to Ex4 and GIP without changes in cAMP or Ca2+ levels. Thus, β-cell SENP1 limits oral glucose intolerance following HFD by ensuring robust insulin secretion at a point downstream of incretin signaling.

Glucagon-like peptide 1 (GLP-1) and cholecystokinin (CCK) are gut-derived peptide hormones known to play important roles in the regulation of gastrointestinal motility and secretion, appetite, and food intake. We have previously demonstrated that both GLP-1 and CCK are produced in the endocrine pancreas of obese mice. Interestingly, while GLP-1 is well known to stimulate insulin secretion by the pancreatic β-cells, direct evidence of CCK promoting insulin release in human islets remains to be determined. Here, we tested whether islet-derived GLP-1 or CCK is necessary for the full stimulation of insulin secretion. We confirm that mouse pancreatic islets secrete GLP-1 and CCK, but only GLP-1 acts locally within the islet to promote insulin release ex vivo. GLP-1 is exclusively produced in approximately 50% of α-cells in lean mouse islets and 70% of α-cells in human islets, suggesting a paracrine α to β-cell signaling through the β-cell GLP-1 receptor. Additionally, we provide evidence that islet CCK expression is regulated by glucose, but its receptor signaling is not required during glucose-stimulated insulin secretion (GSIS). We also see no increase in GSIS in response to CCK peptides. Importantly, all these findings were confirmed in islets from non-diabetic human donors. In summary, our data suggest no direct role for CCK in stimulating insulin secretion and highlight the critical role of intra-islet GLP-1 signaling in the regulation of human β-cell function.

We hypothesize that basal hyperinsulinemia is synergistically mediated by an interplay between increased oxidative stress and excess lipid in the form of reactive oxygen species (ROS) and long-chain acyl-CoA esters (LC-CoA). In addition, ROS production may increase in response to inflammatory cytokines and certain exogenous environmental toxins that mislead β-cells into perceiving nutrient excess when none exists. Thus, basal hyperinsulinemia is envisioned as an adaptation to sustained real or perceived nutrient excess that only manifests as a disease when the excess demand can no longer be met by an overworked β-cell. In this article we will present a testable hypothetical mechanism to explain the role of lipids and ROS in basal hyperinsulinemia and how they differ from glucose-stimulated insulin secretion (GSIS). The model centers on redox regulation, via ROS, and S-acylation–mediated trafficking via LC-CoA. These pathways are well established in neural systems but not β-cells. During GSIS, these signals rise and fall in an oscillatory pattern, together with the other well-established signals derived from glucose metabolism; however, their precise roles have not been defined. We propose that failure to either increase or decrease ROS or LC-CoA appropriately will disturb β-cell function.

The spatial architecture of the islets of Langerhans is hypothesized to facilitate synchronized insulin secretion among β cells, yet testing this in vivo in the intact pancreas is challenging. Robo βKO mice, in which the genes Robo1 and Robo2 are deleted selectively in β cells, provide a unique model of altered islet spatial architecture without loss of β cell differentiation or islet damage from diabetes. Combining Robo βKO mice with intravital microscopy, we show here that Robo βKO islets have reduced synchronized intra-islet Ca2+ oscillations among β cells in vivo. We provide evidence that this loss is not due to a β cell-intrinsic function of Robo, mis-expression or mis-localization of Cx36 gap junctions, or changes in islet vascularization or innervation, suggesting that the islet architecture itself is required for synchronized Ca2+ oscillations. These results have implications for understanding structure-function relationships in the islets during progression to diabetes as well as engineering islets from stem cells.

Pancreatic β-cells couple nutrient metabolism with appropriate insulin secretion. Here, we examined the requirements of pyruvate kinase (PK) isoforms in fuel sensing through the β-cell specific deletion of consititutively active PKM1 and allosterically recruitable PKM2 (PKM1- βKO, PKM2-βKO). β-cell deletion of PEP carboxykinase (PCK2-βKO) was used to block mitochondrial PEP production, leaving glycolytic PEP production intact. In vivo, only the PKM1-βKO mice showed glucose intolerance, and ex vivo insulin secretion was correspondingly reduced. Glucose-dependent Ca2+ oscillations were comparable in PKM1 βKO and PKM2-βKO islets, while the oscillatory frequency and duty cycle in PCK2-βKO islets increased relative to controls, suggesting redundancy in the PK response to glycolytic but not mitochondrial PEP. Using direct single-channel measurements of KATP in intact β-cells, amino acids were sufficient to stimulate KATP closure in control and PKM2-βKO cells, but failed to do so in the PCK2-βKO and PKM1-βKO models. PK activators (PKa) rescued KATP closure in PKM1- but not PCK2-deficient β-cells, arguing that PKM1 and PCK2 mediate membrane depolarization in response to mitochondrial fuels. The dependence of PKM2 on glucose was confirmed by the ability of PKa to rescue KATP closure in excised membrane patches from PKM1-βKO mice. Consistently, calcium influx stimulated by amino acids at low glucose was deficient in islets from PKM1-βKO and PCK2-βKO mice. PKa was sufficient to rescue the calcium defect in the PKM1-βKO islets but did not fully rescue the PCK2-βKO. Our findings provide strong genetic and electrophysiological evidence that the PCK2-PKM1 pathway is uniquely suited to direct the active (secretory) phase of calcium oscillations through direct effects on KATP, when FBP is low and PKM2 is inactive. While more limited in function, PKM2 can be therapeutically targeted, endowing it with the ability to respond to mitochondrial PEP and boost insulin secretion. Disclosure H. R. Foster: None. T. Ho: None. E. Potapenko: None. S. L. Lewandowski: None. S. Sdao: None. H. R. Vandeusen: None. R. L. Cardone: None. R. Kibbey: Research Support; Self; Agios, Inc. M. J. Merrins: None. Funding Health Resources and Services Administration (T32HP10010); National Institute on Aging (T32AG000213); National Institutes of Health (R01DK113103)

Diabetes is characterized by a loss of functional β-cell mass. With current rodent models of chemically-induced diabetes such as streptozotocin, it is difficult to distinguish the consequence of β-cell loss from the decline in individual β-cell function. Here, we generated a mouse model of severe β-cell mass loss by deleting β-cell CDK1 within the first week of life (Cdk1Ucn3Cre, Cdk1-floxed:Ucn3-Cre). At 4 weeks of age, islet mass in Cdk1Ucn3Cre islets is comparable to controls, although islet architecture is disrupted with α-cells having formed more heterotypic contacts with β-cells. By 12 weeks of age, Cdk1Ucn3Cre mice exhibit significantly reduced β-cell mass as well as α-cell mass, but maintain glucose tolerance and ex vivo insulin secretion in response to glucose, amino acids, and the gut incretin GIP. We therefore hypothesized that functional compensations in Cdk1Ucn3Cre islets arise from both β- and α-cells, which are insulinotropic in the fed state. Interestingly, α-cell glucagon secretion from Cdk1Ucn3Cre islets exhibits hypersensitivity to suppression by glucose, including when glucagonotropic amino acids are present. In parallel, we observed a reduction in amino acid-stimulated cAMP in β‑cells from Cdk1Ucn3Cre islets. We also found a left-shift in the glucose dependence of β-cell calcium oscillations in Cdk1Ucn3Cre islets, identifying β-cell recruitment as one mechanism offsetting the loss of α-cell paracrine signaling. Importantly, GIP elicited a much larger glucagon response from glucose- and amino-acid stimulated Cdk1Ucn3Cre islets, implying that α-cell compensation contributes significantly to the maintained insulin secretory output in vivo. In sum, the Cdk1Ucn3Cre model reveals the interdependence of α- and β-cell mass, and function, and will be useful to define the temporal sequence of islet compensation for chronic loss of β-cell mass under stressed conditions. Disclosure S. Sdao: None. M. Adams: None. H. R. Foster: None. B. Blum: None. M. J. Merrins: None.

β-Cell mitochondria play a central role in coupling glucose metabolism with insulin secretion. Here, we identified a metabolic function of cyclin-dependent kinase 1 (CDK1)/cyclin B1—the activation of mitochondrial respiratory complex I—that is active in quiescent adult β-cells and hyperactive in β-cells from obese (ob/ob) mice. In WT islets, respirometry revealed that 65% of complex I flux and 49% of state 3 respiration is sensitive to CDK1 inhibition. Islets from ob/ob mice expressed more cyclin B1 and exhibited a higher sensitivity to CDK1 blockade, which reduced complex I flux by 76% and state 3 respiration by 79%. The ensuing reduction in mitochondrial NADH utilization, measured with two-photon NAD(P)H fluorescence lifetime imaging (FLIM), was matched in the cytosol by a lag in citrate cycling, as shown with a FRET reporter targeted to β-cells. Moreover, time-resolved measurements revealed that in ob/ob islets, where complex I flux dominates respiration, CDK1 inhibition is sufficient to restrict the duty cycle of ATP/ADP and calcium oscillations, the parameter that dynamically encodes β-cell glucose sensing. Direct complex I inhibition with rotenone mimicked the restrictive effects of CDK1 inhibition on mitochondrial respiration, NADH turnover, ATP/ADP, and calcium influx. These findings identify complex I as a critical mediator of obesity-associated metabolic remodeling in β-cells and implicate CDK1 as a regulator of complex I that enhances β-cell glucose sensing.

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.

GIPR activity in α cells is required for the complete metabolic response to a meal., Glucose-dependent insulinotropic polypeptide (GIP) communicates nutrient intake from the gut to islets, enabling optimal levels of insulin secretion via the GIP receptor (GIPR) on β cells. The GIPR is also expressed in α cells, and GIP stimulates glucagon secretion; however, the role of this action in the postprandial state is unknown. Here, we demonstrate that GIP potentiates amino acid–stimulated glucagon secretion, documenting a similar nutrient-dependent action to that described in β cells. Moreover, we demonstrate that GIP activity in α cells contributes to insulin secretion by invoking paracrine α to β cell communication. Last, specific loss of GIPR activity in α cells prevents glucagon secretion in response to a meal stimulus, limiting insulin secretion and driving glucose intolerance. Together, these data uncover an important axis by which GIPR activity in α cells is necessary to coordinate the optimal level of both glucagon and insulin secretion to maintain postprandial homeostasis.

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. Plasma membrane-localized PK is sufficient to close K(ATP) channels and initiate calcium influx. Small-molecule PK activators increase the frequency of ATP/ADP and calcium oscillations and potently amplify insulin secretion. PK restricts respiration by cyclically depriving mitochondria of ADP, which accelerates PEP cycling until membrane depolarization restores ADP and oxidative phosphorylation. Our findings support a compartmentalized model of β-cell metabolism in which PK locally generates the ATP/ADP required for insulin secretion. Oscillatory PK activity allows mitochondria to perform synthetic and oxidative functions without any net impact on glucose oxidation. These findings suggest a potential therapeutic route for diabetes based on PK activation that would not be predicted by the current consensus single-state model of β-cell function.

Of the β-cell signaling pathways altered by non-diabetic obesity and insulin resistance, some are adaptive while others actively contribute to β-cell failure and demise. Cytoplasmic calcium (Ca2+) and cyclic AMP (cAMP), which control the timing and amplitude of insulin secretion, are two important signaling intermediates that can be controlled by stimulatory and inhibitory G protein-coupled receptors. Previous work has shown the importance of the cAMP-inhibitory EP3 receptor in the beta-cell dysfunction of type 2 diabetes. To examine alterations in β-cell cAMP during diabetes progression we utilized a β-cell specific cAMP biosensor in tandem with islet Ca2+recordings and insulin secretion assays. Three groups of C57BL/6J mice were used as a model of the progression from metabolic health to type 2 diabetes: wildtype, normoglycemicLeptinOb, and hyperglycemicLeptinOb. Here, we report robust increases in β-cell cAMP and insulin secretion responses in normoglycemicLeptinobmice as compared to wild-type: an effect that was lost in islets from hyperglycemicLeptinobmice, despite elevated Ca2+duty cycle. Yet, the correlation of EP3 expression and activity to reduce cAMP levels and Ca2+duty cycle with reduced insulin secretion only held true in hyperglycemicLeptinObmice. Our results suggest alterations in beta-cell EP3 signaling may be both adaptive and maladaptive and define β-cell EP3 signaling as much more nuanced than previously understood.

Advances in fluorescent biosensors allow researchers to spatiotemporally monitor a diversity of biochemical reactions and secondary messengers. However, commercial microscopes for the specific application of Förster Resonance Energy Transfer (FRET) are prohibitively expensive to implement in the undergraduate classroom, owing primarily to the dynamic range required and need for ratiometric emission imaging. The purpose of this article is to provide a workflow to design a low-cost, FRET-enabled microscope and to equip the reader with sufficient knowledge to compare commercial light sources, optics, and cameras to modify the device for a specific application. We used this approach to construct a microscope that was assembled by undergraduate students with no prior microscopy experience that is suitable for most single-cell cyan and yellow fluorescent protein FRET applications. The utility of this design was demonstrated by measuring small metabolic oscillations by using a lactate FRET sensor expressed in primary mouse pancreatic islets, highlighting the biologically suitable signal-to-noise ratio and dynamic range of our compact microscope. The instructions in this article provide an effective teaching tool for undergraduate educators and students interested in implementing FRET in a cost-effective manner.

SummaryThe mitochondrial GTP (mtGTP)-dependent phosphoenolpyruvate (PEP) cycle is an anaplerotic-cataplerotic mitochondrial shuttle utilizing mitochondrial PEPCK (PCK2) and pyruvate kinase (PK). PEP cycling stimulates insulin secretion via OxPhos-independent lowering of ADP by PK. We assess in vivo whether islet PCK2 is necessary for glucose sensing and if speeding the PEP cycle via pharmacological PK activators amplifies insulin secretion. Pck2-/- mice had severely impaired insulin secretion during islet perifusion, oral glucose tolerance tests and hyperglycemic clamps. Acute and chronic pharmacologic PK activator therapy improved islet insulin secretion from normal, high-fat diet (HFD) fed, or Zucker diabetic fatty (ZDF) rats, and glucolipotoxic or diabetic humans. A similar improvement in insulin secretion was observed in regular chow and HFD rats in vivo. Insulin secretion and cytosolic Ca2+ during PK activation were dependent on PCK2. These data provide a preclinical rationale for strategies, such as PK activation, that target the PEP cycle to improve glucose homeostasis.HighlightsLoss of mitochondrial phosphoenolpyruvate (PEP) impairs insulin release in vivo.Pyruvate kinase (PK) activators stimulate beta-cells in preclinical diabetes models.PEP cycling in vivo depends on PK and mitochondrial PEPCK (PCK2) for insulin release.Acute and 3-week oral PK activator amplifies insulin release during hyperglycemia.eTOC BlurbAbudukadier et al. show that small molecule pyruvate kinase activation in vivo and in vitro increases insulin secretion in rodent and human models of diabetes. The phosphoenolpyruvate (PEP) cycling mechanism and its amplification are dependent on mitochondrial PEPCK (PCK2).

SUMMARYPancreatic 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 KATPchannels 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.GRAPHICAL ABSTRACTThe consensus model for β-cell glucose sensing supports a dominant role for OxPhos. This model doesn’t fully explain the observed metabolic and electrophysiologic oscillations associated with glucose-stimulated insulin secretion. Lewandowskiet al. challenge this model by mechanistically connecting the anaplerotic PEP cycle to the electrically silent triggering phase, and OxPhos to the electrically active secretory phase. Here, the allosteric recruitment of pyruvate kinase directs metabolic traffic between the two cycles and identifies potential therapeutic strategies for diabetes based on pharmacologic pyruvate kinase activation.HIGHLIGHTSCompartmentalized pyruvate kinase (PK) activity underlies β-cell fuel sensingMembrane-associated PK closes KATPchannels and controls calcium influxBy lowering ADP, PK toggles mitochondria between OxPhos and PEP biosynthesisPharmacologic PK activation increases oscillatory frequency and amplifies secretion

The spatial architecture of the islets of Langerhans is hypothesized to facilitate synchronized insulin secretion between β cells, yet testing this in vivo in the intact pancreas is challenging. Robo βKO mice, in which the genes Robo1 and Robo2 are deleted selectively in β cells, provide a unique model of altered islet architecture without loss of β cell differentiation or islet damage from diabetes. Combining Robo βKO mice with intravital microscopy, we show here that Robo βKO islets lose synchronized intra-islet Ca2+ oscillations between β cells in vivo. We provide evidence that this loss is not due to a β cell-intrinsic function of Robo, loss of Connexin36 gap junctions, or changes in islet vascularization, suggesting that the islet architecture itself is required for synchronized Ca2+ oscillations. These results will have implications for understanding structure-function relationships in the islets during progression to diabetes as well as engineering islets from stem cells.

The spatial architecture of the islets of Langerhans is hypothesized to facilitate synchronized insulin secretion between β cells, yet testing thisin vivoin the intact pancreas is challenging.Robo βKOmice, in which the genesRobo1andRobo2are deleted selectively in β cells, provide a unique model of altered islet spatial architecture without loss of β cell differentiation or islet damage from diabetes. CombiningRobo βKOmice with intravital microscopy, we show here thatRobo βKOislets lose synchronized intra-islet Ca2+oscillations between β cellsin vivo. We provide evidence that this loss is not due to a β cell-intrinsic function of Robo, loss of Connexin36 gap junctions, or changes in islet vascularization, suggesting that the islet architecture itself is required for synchronized Ca2+oscillations. These results have implications for understanding structure-function relationships in the islets during progression to diabetes as well as engineering islets from stem cells.

While NAD(P)H fluorescence lifetime imaging (FLIM) can detect changes in flux through the TCA cycle and electron transport chain (ETC), it remains unclear whether NAD(P)H FLIM is sensitive to other potential fates of glucose. Glucose carbon can be diverted from mitochondria by the pentose phosphate pathway (via glucose 6-phosphate dehydrogenase, G6PDH), lactate production (via lactate dehydrogenase, LDH), and rejection of carbon from the TCA cycle (via pyruvate dehydrogenase kinase, PDK), all of which can be upregulated in cancer cells. Here, we demonstrate that multiphoton NAD(P)H FLIM can be used to quantify the relative concentrations of recombinant LDH and malate dehydrogenase (MDH) in solution. In multiple epithelial cell lines, NAD(P)H FLIM was also sensitive to inhibition of LDH and PDK, as well as the directionality of LDH in cells forced to use pyruvate versus lactate as fuel sources. Among the parameters measurable by FLIM, only the lifetime of protein-bound NAD(P)H (τ2) was sensitive to these changes, in contrast to the optical redox ratio, mean NAD(P)H lifetime, free NAD(P)H lifetime, or the relative amount of free and protein-bound NAD(P)H. NAD(P)H τ2 offers the ability to non-invasively quantify diversions of carbon away from the TCA cycle/ETC, which may support mechanisms of drug resistance.

Paracrine interactions between pancreatic islet cells have been proposed as a mechanism to regulate hormone secretion and glucose homeostasis. Here, we demonstrate the importance of proglucagon-derived peptides (PGDPs) for α to β cell communication and control of insulin secretion. Signaling through this system occurs through both the glucagon-like peptide receptor (Glp1r) and glucagon receptor (Gcgr). Loss of PGDPs, or blockade of their receptors, decreases insulin secretion in response to both metabolic and nonmetabolic stimulation of mouse and human islets. This effect is due to reduced β cell cAMP and affects the quantity but not dynamics of insulin release, indicating that PGDPs dictate the magnitude of insulin output in an isolated islet. In healthy mice, additional factors that stimulate cAMP can compensate for loss of PGDP signaling; however, input from α cells is essential to maintain glucose tolerance during the metabolic stress induced by high-fat feeding. These findings demonstrate an essential role for α cell regulation of β cells, raising the possibility that abnormal paracrine signaling contributes to impaired insulin secretion in diabetes. Moreover, these findings support reconsideration of the role for α cells in postprandial glucose control.

Pancreatic islets respond to elevated blood glucose by secreting pulses of insulin that parallel oscillations in β-cell metabolism, intracellular Ca(2+) concentration, and bursting electrical activity. The mechanisms that maintain an oscillatory response are not fully understood, yet several models have been proposed. Only some can account for experiments supporting that metabolism is intrinsically oscillatory in β-cells. The dual oscillator model (DOM) implicates glycolysis as the source of oscillatory metabolism. In the companion article, we use recently developed biosensors to confirm that glycolysis is oscillatory and further elucidate the coordination of metabolic and electrical signals in the insulin secretory pathway. In this report, we modify the DOM by incorporating an established link between metabolism and intracellular Ca(2+) to reconcile model predictions with experimental observations from the companion article. With modification, we maintain the distinguishing feature of the DOM, oscillatory glycolysis, but introduce the ability of Ca(2+) influx to reshape glycolytic oscillations by promoting glycolytic efflux. We use the modified model to explain measurements from the companion article and from previously published experiments with islets.

Summary The mitochondrial GTP (mtGTP)-dependent phosphoenolpyruvate (PEP) cycle couples mitochondrial PEPCK (PCK2) to pyruvate kinase (PK) in the liver and pancreatic islets to regulate glucose homeostasis. Here, small molecule PK activators accelerated the PEP cycle to improve islet function, as well as metabolic homeostasis, in preclinical rodent models of diabetes. In contrast, treatment with a PK activator did not improve insulin secretion in pck2−/− mice. Unlike other clinical secretagogues, PK activation enhanced insulin secretion but also had higher insulin content and markers of differentiation. In addition to improving insulin secretion, acute PK activation short-circuited gluconeogenesis to reduce endogenous glucose production while accelerating red blood cell glucose turnover. Four-week delivery of a PK activator in vivo remodeled PK phosphorylation, reduced liver fat, and improved hepatic and peripheral insulin sensitivity in HFD-fed rats. These data provide a preclinical rationale for PK activation to accelerate the PEP cycle to improve metabolic homeostasis and insulin sensitivity.

In pancreatic β-cells, glucose metabolism generates signals that trigger and amplify insulin secretion. We generated β-cell specific FRET biosensors for the real-time measurement of cytosolic citrate and glutamate, two putative metabolic coupling factors with an undefined causal relationship to the signaling process. In islet β-cells, glucose elevation from 2.5 to 10 mM was accompanied by a sharp decrease in cytosolic citrate and glutamate. Steady-state oscillations in these intermediates induced by 10 mM glucose reveal that cytosolic citrate and glutamate levels decrease as calcium levels increase, precisely when the TCA cycle is most active. Subsequent membrane depolarization with 30 mM KCl resulted in a further decrease in citrate and glutamate, suggesting that mitochondria respond to work in the form of ATP hydrolysis by importing citrate and glutamate into the TCA cycle. Indeed, parallel measurements with Perceval-HR biosensors show that cytosolic ADP/ATP rises with calcium, and corresponds to a burst of mitochondrial NADH fluorescence. These data are inconsistent with existing models of the amplifying pathway in which mitochondrial glucose oxidation results in the export of TCA intermediates to the cytosol to generate NADPH and GSH/GSSG. The observed increase in cytosolic GSH/GSSG in response to glucose, shown with a FRET reporter targeted to β-cells, implies that GSH is generated from other sources such as the pentose phosphate pathway. Our results support the strong prediction that glucose oxidation in the mitochondria is inversely proportional to insulin secretion via the NADPH-generating citrate and glutamate cycles in the cytosol, since these pathways can never occur simultaneously. Disclosure S.L. Lewandowski: None. M.J. Merrins: None.

The loss of insulin secretion is a hallmark of diabetes, and it is becoming clear that cell cycle regulators control insulin secretion as well as proliferation. It was recently reported that embryonic deletion of cyclin-dependent kinase 2 (Cdk2) in β-cells (Pdx1-Cre:Cdk2-/-) results in impaired glucose tolerance due to defective insulin secretion (Kim et al., J Biol Chem, 2017), revealing a developmental or perinatal requirement of Cdk2. Here, we show that tamoxifen-inducible deletion of Cdk2 in adult β-cells (MIP-CreER:Cdk2-/-) enhances insulin secretion. Despite these differences, both models of Cdk2 depletion exhibited a strong increase in the glucose dependence of β-cell calcium oscillations. This effect is likely due to the loss of ATP-sensitive K+ (KATP) channels, since kir6.2 transcript was reduced in Cdk2-null β-cells, and pharmacological inhibition of Cdk2 correspondingly reduced KATP channel conductance. These results reveal that Cdk2 inhibits insulin secretion by limiting plasma membrane excitability via KATP. In adult β-cells lacking Cdk2, we also observed a 45% reduction in depolarization-induced exocytosis, indicating that Cdk2 is required for the metabolic amplification of insulin secretion. Within the intermediary metabolic pathways, Cdk2-null β-cells exhibited reductions in glucose-dependent lactate accumulation, mitochondrial membrane potential, and mitochondrial NADH utilization. Together, these studies reveal that Cdk2 transcriptionally limits β-cell membrane excitability and the insulin secretory response, and is required to maintain proper metabolic function. Disclosure S. Sdao: None. C. Poudel: None. K.M. Mortensen: None. M.J. Merrins: None.

While NAD(P)H fluorescence lifetime imaging (FLIM) can detect changes in flux through the TCA cycle and electron transport chain (ETC), it remains unclear whether NAD(P)H FLIM is sensitive to other potential fates of glucose. Glucose carbon can be diverted from mitochondria by the pentose phosphate pathway (via glucose 6-phosphate dehydrogenase, G6PDH), lactate production (via lactate dehydrogenase, LDH), and rejection of carbon from the TCA cycle (via pyruvate dehydrogenase kinase, PDK), all of which can be upregulated in cancer cells. Here, we demonstrate that NAD(P)H FLIM can be used to quantify the relative concentrations of recombinant LDH and malate dehydrogenase (MDH) in solution. In multiple epithelial cell lines, NAD(P)H FLIM was also sensitive to inhibition of LDH and PDK, as well as the directionality of LDH in cells forced to use pyruvate versus lactate as a fuel source. Among the parameters measurable by FLIM, only the lifetime of protein-bound NAD(P)H (τ_2) was sensitive to these changes, in contrast to the optical redox ratio, mean NAD(P)H lifetime, free NAD(P)H lifetime, or the relative amount of free and protein-bound NAD(P)H. NAD(P)H τ_2 offers the ability to non-invasively quantify diversions of carbon away from the TCA cycle/ETC, which may support mechanisms of drug resistance. It remains unclear whether NAD(P)H FLIM is sensitive to potential fates of glucose other than the TCA cycle and electron transport chain. We show that the lifetime of protein-bound NAD(P)H (τ_2) can distinguish the relative concentrations of malate dehydrogenase (TCA cycle enzyme) and lactate dehydrogenase (diverts carbon to/from lactate production) in solutions containing both enzymes. In cells, NAD(P)H FLIM was also sensitive to alterations in the path of carbon from glucose uptake to mitochondrial activity. Additionally, τ_2 was found to be the FLIM parameter best-suited for detecting these carbon-diverting shifts in cell metabolism, which may support mechanisms of drug resistance.

The noninvasive measurement of functional β-cell mass would be clinically valuable for monitoring the progression of type 1 and type 2 diabetes, as well as the viability of transplanted insulin-producing cells. Although previous work employing magnetic resonance imaging has shown promise for functional β-cell mass determination through voltage-dependent Ca2+ channel (VDCC)-mediated internalization of Mn2+, the clinical utility of this technique is limited by the cytotoxic levels of Mn2+ contrast agent. Here, we show that positron emission tomography (PET) is advantageous for determining functional β-cell mass using 52Mn2+ (t1/2: 5.6 d). We investigated the whole-body distribution of 52Mn2+ in healthy adult mice by dynamic and static PET imaging. Pancreatic VDCC uptake of 52Mn2+ was successfully manipulated pharmacologically in vitro and in vivo using glucose, nifedipine (VDCC blocker), the sulfonylureas tolbutamide and glibenclamide (KATP channel blockers), and diazoxide (KATP channel opener). In a mouse model of streptozotocin (STZ)-induced type 1 diabetes, 52Mn2+ uptake in the pancreas was distinguished from healthy controls in parallel with classic histological quantification of β-cell mass from pancreatic sections. 52Mn2+-PET also reported the expected increase in functional β-cell mass in the ob/ob model of pre-type 2 diabetes, a result corroborated by histological β-cell mass measurements and live-cell imaging of β-cell Ca2+ oscillations. These results indicate that 52Mn2+-PET is a sensitive new tool for the non-invasive assessment of functional β-cell mass.

Prostaglandin E2 (PGE2) is derived from arachidonic acid, whereas PGE3 is derived from eicosapentaenoic acid (EPA) using the same downstream metabolic enzymes. Little is known about the impact of EPA and PGE3 on β-cell function, particularly in the diabetic state. In this work, we determined that PGE3 elicits a 10-fold weaker reduction in glucose-stimulated insulin secretion through the EP3 receptor as compared with PGE2. We tested the hypothesis that enriching pancreatic islet cell membranes with EPA, thereby reducing arachidonic acid abundance, would positively impact β-cell function in the diabetic state. EPA-enriched islets isolated from diabetic BTBR Leptinob/ob mice produced significantly less PGE2 and more PGE3 than controls, correlating with improved glucose-stimulated insulin secretion. NAD(P)H fluorescence lifetime imaging showed that EPA acts downstream and independently of mitochondrial function. EPA treatment also reduced islet interleukin-1β expression, a proinflammatory cytokine known to stimulate prostaglandin production and EP3 expression. Finally, EPA feeding improved glucose tolerance and β-cell function in a mouse model of diabetes that incorporates a strong immune phenotype: the NOD mouse. In sum, increasing pancreatic islet EPA abundance improves diabetic β-cell function through both direct and indirect mechanisms that converge on reduced EP3 signaling.

Protein-restricted (PR), high-carbohydrate diets improve metabolic health in rodents, yet the precise dietary components that are responsible for these effects have not been identified. Furthermore, the applicability of these studies to humans is unclear. Here, we demonstrate in a randomized controlled trial that a moderate PR diet also improves markers of metabolic health in humans. Intriguingly, we find that feeding mice a diet specifically reduced in branched-chain amino acids (BCAAs) is sufficient to improve glucose tolerance and body composition equivalently to a PR diet via metabolically distinct pathways. Our results highlight a critical role for dietary quality at the level of amino acids in the maintenance of metabolic health and suggest that diets specifically reduced in BCAAs, or pharmacological interventions in this pathway, may offer a translatable way to achieve many of the metabolic benefits of a PR diet. © 2016 The Authors

Dysregulation of endoplasmic reticulum (ER) homeostasis contributes to β-cell dysfunction, but it remains unclear how the dynamics of metabolic oscillations are affected. Using both fitting and phasor analyses of NAD(P)H lifetimes, we observed an increase in the lifetime of bound NAD(P)H in islets harvested from ob/ob mice, an in vivo model of obesity and ER stress, relative to wild-type controls. The downstream effects of mitochondrial-driven cellular dynamics were quantified using combinations of FRET biosensors, fluorescent dyes, and genetically-encoded reporters. Analysis of the oscillations revealed that ER stress modulates the dynamics of mitochondrial NAD(P)H and membrane potential (Δψm), ATP/ADP, and cytosolic and ER Ca2+. In ob/ob islets, the β-cell's sensitivity to glucose was fundamentally increased, as evidenced by a ∼3-fold increase in oscillatory plateau fraction and augmented insulin exocytosis. In vivo administration of the chemical ER stress mitigator tauroursodeoxycholic acid (TUDCA) - currently in Phase I Clinical trials - rescued the oscillation dynamics in ob/ob islets by relaxing β-cell glucose sensitivity and reducing baseline levels of cytosolic Ca2+. Reduction of β-cell excitability may improve long term survival in obese animals.

Pancreatic islets exhibit bursting oscillations in response to elevated blood glucose. These oscillations are accompanied by oscillations in the free cytosolic Ca 2+ concentration (Cac), which drives pulses of insulin secretion. Both islet Ca 2+ and metabolism oscillate, but there is some debate about their interrelationship. Recent experimental data show that metabolic oscillations in some cases persist after the addition of diazoxide (Dz), which opens K(ATP) channels, hyperpolarizing β- cells and preventing Ca 2+ entry and Ca 2+ oscillations. Further, in some islets in which metabolic oscillations were eliminated with Dz, increasing the cytosolic Ca 2+ concentration by the addition of KCl could restart the metabolic oscillations. Here we address why metabolic oscillations persist in some islets but not others, and why raising Cac restarts oscillations in some islets but not others. We answer these questions using the dual oscillator model (DOM) for pancreatic islets. The DOM can reproduce the experimental data and shows that the model supports two different mechanisms for slow metabolic oscillations, one that requires calcium oscillations and one that does not.

Pulses of insulin released from pancreatic β-cells maintain blood glucose in a narrow range, although the source of these pulses is unclear. We and others have proposed that positive feedback mediated by the glycolytic enzyme phosphofructokinase-1 (PFK1) enables β-cells to generate metabolic oscillations via autocatalytic activation by its product fructose 1,6-bisphosphate (FBP). Although much indirect evidence has accumulated in favor of this hypothesis, a direct measurement of oscillating glycolytic intermediates has been lacking. To probe glycolysis directly, we engineered a family of inter- and intramolecular FRET biosensors based on the glycolytic enzyme pyruvate kinase M2 (PKAR; pyruvate kinase activity reporter), which multimerizes and is activated upon binding FBP. When introduced into Min6 β-cells, PKAR FRET efficiency increased rapidly in response to glucose. Importantly, however, metabolites entering downstream of PFK1 (glyceraldehyde, pyruvate, and ketoisocaproate) failed to activate PKAR, consistent with sensor activation by FBP; the dependence of PKAR on FBP was further confirmed using purified sensor in vitro. Using a novel imaging modality for monitoring mitochondrial flavin fluorescence in mouse islets, we show that slow oscillations in mitochondrial redox potential stimulated by 10 mm glucose are in phase with glycolytic efflux through PKM2, measured simultaneously from neighboring islet β-cells expressing PKAR. These results indicate that PKM2 activity in β-cells is oscillatory and are consistent with pulsatile PFK1 being the mediator of slow glycolytic oscillations.

Pulses of insulin from pancreatic beta-cells help maintain blood glucose in a narrow range, although the source of these pulses is unclear. It has been proposed that a positive feedback circuit exists within the glycolytic pathway, the autocatalytic activation of phosphofructokinase-1 (PFK1), which endows pancreatic beta-cells with the ability to generate oscillations in metabolism. Flux through PFK1 is controlled by the bifunctional enzyme PFK2/FBPase2 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase) in two ways: via (1) production/degradation of fructose-2,6-bisphosphate (Fru2,6-BP), a potent allosteric activator of PFK1, as well as (2) direct activation of glucokinase due to a protein-protein interaction. In this study, we used a combination of live-cell imaging and mathematical modeling to examine the effects of inducibly-expressed PFK2/FBPase2 mutants on glucose-induced Ca(2+) pulsatility in mouse islets. Irrespective of the ability to bind glucokinase, mutants of PFK2/FBPase2 that increased the kinase:phosphatase ratio reduced the period and amplitude of Ca(2+) oscillations. Mutants which reduced the kinase:phosphatase ratio had the opposite effect. These results indicate that the main effect of the bifunctional enzyme on islet pulsatility is due to Fru2,6-BP alteration of the threshold for autocatalytic activation of PFK1 by Fru1,6-BP. Using computational models based on PFK1-generated islet oscillations, we then illustrated how moderate elevation of Fru-2,6-BP can increase the frequency of glycolytic oscillations while reducing their amplitude, with sufficiently high activation resulting in termination of slow oscillations. The concordance we observed between PFK2/FBPase2-induced modulation of islet oscillations and the models of PFK1-driven oscillations furthermore suggests that metabolic oscillations, like those found in yeast and skeletal muscle, are shaped early in glycolysis.

Extended abstract of a paper presented at Microscopy and Microanalysis 2011 in Nashville, Tennessee, USA, August 7–August 11, 2011.

Pulses of insulin from pancreatic beta-cells help maintain blood glucose in a narrow range, although the source of these pulses is unclear. We propose that a positive feedback circuit exists within the glycolytic pathway employing the allosteric enzyme phosphofructokinase-1 (PFK1), which endows beta-cells with the ability to generate oscillations in metabolism via autocatalytic activation by its product Fructose-1,6-bisphosphate (Fru1,6-BP). To test this hypothesis, we have engineered a family of inter- and intramolecular Cerulean/Citrine FRET biosensors based on the glycolytic enzyme pyruvate kinase M2 (PK), which is allosterically activated and reported to multimerize upon binding Fru1,6-BP. When introduced into Min6 beta-cells, intramolecular PK biosensor apparent FRET efficiency increased dose-dependently in response to glucose (0, 2.5, 11, and 25 mM). This change was rapid (within seconds) and reversible. When Min6 cells were stimulated with 25 mM glucose/TEA, oscillations in PK biosensor activity were evident as ratiometric FRET changes, and exhibited a similar period to slow oscillations in intracellular calcium or NAD(P)H (3-4 min). Our results suggest that glycolysis in beta-cells is oscillatory and that PFK1 is indeed an attractive candidate for the oscillatory generator. More broadly, this family of PK biosensor constructs could be useful for exploring the magnitude and kinetics of glycolytic activity in living cells. Supported by F32DK085960 (M.J.M.) and R01DK46409 (L.S.).