Insulin secretion deficits in a Prader-Willi syndrome ß-cell model are associated with a concerted downregulation of multiple endoplasmic reticulum chaperones.

Prader-Willi syndrome (PWS) is a multisystem disorder with neurobehavioral, metabolic, and hormonal phenotypes, caused by loss of expression of a paternally-expressed imprinted gene cluster. Prior evidence from a PWS mouse model identified abnormal pancreatic islet development with retention of aged insulin and deficient insulin secretion. To determine the collective roles of PWS genes in ß-cell biology, we used genome-editing to generate isogenic, clonal INS-1 insulinoma lines having 3.16 Mb deletions of the silent, maternal- (control) and active, paternal-allele (PWS). PWS ß-cells demonstrated a significant cell autonomous reduction in basal and glucose-stimulated insulin secretion. Further, proteomic analyses revealed reduced levels of cellular and secreted hormones, including all insulin peptides and amylin, concomitant with reduction of at least ten endoplasmic reticulum (ER) chaperones, including GRP78 and GRP94. Critically, differentially expressed genes identified by whole transcriptome studies included reductions in levels of mRNAs encoding these secreted peptides and the group of ER chaperones. In contrast to the dosage compensation previously seen for ER chaperones in Grp78 or Grp94 gene knockouts or knockdown, compensation is precluded by the stress-independent deficiency of ER chaperones in PWS ß-cells. Consistent with reduced ER chaperones levels, PWS INS-1 ß-cells are more sensitive to ER stress, leading to earlier activation of all three arms of the unfolded protein response. Combined, the findings suggest that a chronic shortage of ER chaperones in PWS ß-cells leads to a deficiency of protein folding and/or delay in ER transit of insulin and other cargo. In summary, our results illuminate the pathophysiological basis of pancreatic ß-cell hormone deficits in PWS, with evolutionary implications for the multigenic PWS-domain, and indicate that PWS-imprinted genes coordinate concerted regulation of ER chaperone biosynthesis and ß-cell secretory pathway function.

Riding the rapids: COVID-19, the three rivers curriculum, and the experiences of the University of Pittsburgh School of Medicine.

When faced with the COVID-19 pandemic this past spring, the University of Pittsburgh's School of Medicine (UPSOM) took rapid steps to ensure the safety of students, staff, and the faculty as well as to maintain the educational process. Curriculum reform efforts, already underway, proved to be an advantage in the transformation. We quickly converted to a remote and then a hybrid curriculum. Research labs were reopened with appropriate safety measures. Clinical experiences for students restarted via a phased process that emphasized safety and graduation requirements. A variety of assessment mechanisms were restarted with appropriate modifications. New teaching models, such as flipped classrooms, have become the norm, and it seems hard to imagine our returning to our old pedagogy. The curriculum committee met continually to guide the process of change and reopening. The curricular adaptation process remains ongoing, and challenges remain. Nonetheless, we have learned from our experiences and hope to use this knowledge gained as we move forward.

Autocrine C-peptide protects INS1 ß cells against palmitic acid-induced oxidative stress in peroxisomes by inducing catalase.

AIMS: C-peptide, produced by pancreatic ß cells and co-secreted in the bloodstream with insulin, has antioxidant properties in glucose- and hydrogen peroxide (H2O2)-exposed INS1 ß cells. Palmitic acid, the most physiologically abundant long-chain free fatty acid in humans, is metabolized in peroxisomes of ß cells accumulating H2O2 that can lead to oxidative stress. Here, we tested the hypothesis that C-peptide protects ß cells from palmitic acid-induced stress by lowering peroxisomal H2O2. MATERIALS AND METHODS: We exposed INS1 ß cells to palmitic acid and C-peptide in the setting of increasing glucose concentration and tested for changes in parameters of stress and death. To study the ability of C-peptide to lower peroxisomal H2O2, we engineered an INS1 ß cell line stably expressing the peroxisomal-targeted H2O2 sensor HyPer, whose fluorescence increases with cellular H2O2. An INS1 ß cell line stably expressing a live-cell fluorescent catalase reporter was used to detect changes in catalase gene expression. RESULTS: C-peptide protects INS1 ß cells from the combined effect of palmitic acid and glucose by reducing peroxisomal H2O2 to baseline levels and increasing expression of catalase. CONCLUSIONS: In conditions of glucolipotoxicity, C-peptide increases catalase expression and reduces peroxisomal oxidative stress and death of INS1 ß cells. Maintenance of C-peptide secretion is a pro-survival requisite for ß cells in adverse conditions. Loss of C-peptide secretion would render ß cells more vulnerable to stress and death leading to secretory dysfunction and diabetes.

Glucose but not KCl diminishes submembrane granule turnover in mouse beta-cells.

KCl depolarization is widely used to mimic the depolarization during glucose-stimulated insulin secretion. Consequently, the insulin secretion elicited by KCl is often regarded as the equivalent of the first phase of glucose-induced insulin secretion. Here, the effects of both stimuli were compared by measuring the secretion of perifused mouse islets, the cytosolic Ca2+ concentration of single beta-cells and the mobility of submembrane insulin granules by TIRF microscopy of primary mouse beta-cells. Two cargo-directed granule labels were used namely insulin-EGFP and C-peptide-emGFP. The granule behaviour common to both was used to compare the effect of sequential stimulation with 40 mM KCl and 30 mM glucose and sequential stimulation with the same stimuli in reversed order. At the level of the cell secretory response, the sequential pulse protocol showed marked differences depending on the order of the two stimuli. KCl produced higher maximal secretion rates and diminished the response to the subsequent glucose stimulus, whereas glucose enhanced the response to the subsequent KCl stimulus. At the level of granule behaviour, a difference developed during the first stimulation phase in that the total number of granules, the short-term resident granules and the arriving granules, which are all parameters of granule turnover, were significantly smaller for glucose than for KCl. These differences at both the level of the cell secretory response and granule behaviour in the submembrane space are incompatible with identical initial response mechanisms to KCl and glucose stimulation.

Autocrine C-peptide mechanism underlying INS1 beta cell adaptation to oxidative stress.

BACKGROUND: Excessive generation of reactive oxygen species (ROS) causing oxidative stress plays a major role in the pathogenesis of diabetes by inducing beta cell secretory dysfunction and apoptosis. Recent evidence has shown that C-peptide, produced by beta cells and co-secreted with insulin in the circulation of healthy individuals, decreases ROS and prevents apoptosis in dysfunctional vascular endothelial cells. In this study, we tested the hypothesis that an autocrine activity of C-peptide similarly decreases ROS when INS1 beta cells are exposed to stressful conditions of diabetes. METHODS: Reactive oxygen species and apoptosis were induced in INS1 beta cells pretreated with C-peptide by either 22 mM glucose or 100 µM hydrogen peroxide (H2 O2 ). To test C-peptide's autocrine activity, endogenous C-peptide secretion was inhibited by the KATP channel opener diazoxide and H2 O2 -induced ROS assayed after addition of either exogenous C-peptide or the secretagogue glibenclamide. In similar experiments, extracellular potassium, which depolarizes the membrane otherwise hyperpolarized by diazoxide, was used to induce endogenous C-peptide secretion. ROS was measured using the cell-permeant dye chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H2 -DCFDA). Insulin secretion and apoptosis were assayed by enzyme-linked immunosorbent assay. RESULTS: C-peptide significantly decreased high glucose-induced and H2 O2 -induced ROS and prevented apoptosis of INS1 beta cells. Diazoxide significantly increased H2 O2 -induced ROS, which was reversed by exogenous C-peptide or glibenclamide or potassium chloride. CONCLUSIONS: These findings demonstrate an autocrine C-peptide mechanism in which C-peptide is bioactive on INS1 beta cells exposed to stressful conditions and might function as a natural antioxidant to limit beta cell dysfunction and loss contributing to diabetes.

Differential regulation and localization of carboxypeptidase D and carboxypeptidase E in human and mouse ß-cells.

Hyperglycemia can result from a relative or absolute lack of functional insulin secreted by the pancreatic ß-cells. Prohormone processing enzymes play an essential role in the secretion of mature and fully functional insulin. Defects in insulin processing enzymes including prohormone convertases 1/3 and 2, and carboxypeptidase E (CPE) can lead to ß-cell stress and hyperproinsulinemia, both of which are features of type 2 diabetes. Despite their importance, the regulation and role of this family of enzymes remain to be fully elucidated. Previously, we demonstrated that lipotoxicity led to the degradation of CPE, but did not affect its related enzyme, carboxypeptidase D (CPD). In this study, we found that CPD was significantly up-regulated by elevated glucose, while CPE was not. Low doses of insulin also increased CPD protein levels, consistent with a role for autocrine signaling. Glucose and insulin did not affect CPD or CPE expression in an α-cell line. Furthermore, insulin treatment altered the CPD sub-cellular localization, which was distinct from CPE. Somewhat surprisingly, the loss of CPE did not affect the levels of CPD. Knockdown of CPD exerted no effect on CPE protein levels. In addition, while our previous study demonstrated that even modest reduction of CPE was sufficient to induce ß-cell apoptosis, CPD knockdown did not affect cell viability. Taken together, our data demonstrate that CPE and CPD are differentially localized, differentially regulated and unlikely to have compensatory functions in pancreatic ß-cells.

Global deficits in development, function, and gene expression in the endocrine pancreas in a deletion mouse model of Prader-Willi syndrome.

Prader-Willi syndrome (PWS) is a multisystem disorder caused by genetic loss of function of a cluster of imprinted, paternally expressed genes. Neonatal failure to thrive in PWS is followed by childhood-onset hyperphagia and obesity among other endocrine and behavioral abnormalities. PWS is typically assumed to be caused by an unknown hypothalamic-pituitary dysfunction, but the underlying pathogenesis remains unknown. A transgenic deletion mouse model (TgPWS) has severe failure to thrive, with very low levels of plasma insulin and glucagon in fetal and neonatal life prior to and following onset of progressive hypoglycemia. In this study, we tested the hypothesis that primary deficits in pancreatic islet development or function may play a fundamental role in the TgPWS neonatal phenotype. Major pancreatic islet hormones (insulin, glucagon) were decreased in TgPWS mice, consistent with plasma levels. Immunohistochemical analysis of the pancreas demonstrated disrupted morphology of TgPWS islets, with reduced α- and ß-cell mass arising from an increase in apoptosis. Furthermore, in vivo and in vitro studies show that the rate of insulin secretion is significantly impaired in TgPWS ß-cells. In TgPWS pancreas, mRNA levels for genes encoding all pancreatic hormones, other secretory factors, and the ISL1 transcription factor are upregulated by either a compensatory response to plasma hormone deficiencies or a primary effect of a deleted gene. Our findings identify a cluster of imprinted genes required for the development, survival, coordinate regulation of genes encoding hormones, and secretory function of pancreatic endocrine cells, which may underlie the neonatal phenotype of the TgPWS mouse model.

α-Synuclein binds the K(ATP) channel at insulin-secretory granules and inhibits insulin secretion.

α-Synuclein has been studied in numerous cell types often associated with secretory processes. In pancreatic ß-cells, α-synuclein might therefore play a similar role by interacting with organelles involved in insulin secretion. We tested for α-synuclein localizing to insulin-secretory granules and characterized its role in glucose-stimulated insulin secretion. Immunohistochemistry and fluorescent sulfonylureas were used to test for α-synuclein localization to insulin granules in ß-cells, immunoprecipitation with Western blot analysis for interaction between α-synuclein and K(ATP) channels, and ELISA assays for the effect of altering α-synuclein expression up or down on insulin secretion in INS1 cells or mouse islets, respectively. Differences in cellular phenotype between α-synuclein knockout and wild-type ß-cells were found by using confocal microscopy to image the fluorescent insulin biosensor Ins-C-emGFP and by using transmission electron microscopy. The results show that anti-α-synuclein antibodies labeled secretory organelles within ß-cells. Anti-α-synuclein antibodies colocalized with K(ATP) channel, anti-insulin, and anti-C-peptide antibodies. α-Synuclein coimmunoprecipitated in complexes with K(ATP) channels. Expression of α-synuclein downregulated insulin secretion at 2.8 mM glucose with little effect following 16.7 mM glucose stimulation. α-Synuclein knockout islets upregulated insulin secretion at 2.8 and 8.4 mM but not 16.7 mM glucose, consistent with the depleted insulin granule density at the ß-cell surface membranes observed in these islets. These findings demonstrate that α-synuclein interacts with K(ATP) channels and insulin-secretory granules and functionally acts as a brake on secretion that glucose stimulation can override. α-Synuclein might play similar roles in diabetes as it does in other degenerative diseases, including Alzheimer's and Parkinson's diseases.

Antidiabetic sulfonylurea stimulates insulin secretion independently of plasma membrane KATP channels.

Understanding mechanisms by which glibenclamide stimulates insulin release is important, particularly given recent promising treatment by glibenclamide of permanent neonatal diabetic subjects. Antidiabetic sulfonylureas are thought to stimulate insulin secretion solely by inhibiting their high-affinity ATP-sensitive potassium (K(ATP)) channel receptors at the plasma membrane of beta-cells. This normally occurs during glucose stimulation, where ATP inhibition of plasmalemmal K(ATP) channels leads to voltage activation of L-type calcium channels for rapidly switching on and off calcium influx, governing the duration of insulin secretion. However, growing evidence indicates that sulfonylureas, including glibenclamide, have additional K(ATP) channel receptors within beta-cells at insulin granules. We tested nonpermeabilized beta-cells in mouse islets for glibenclamide-stimulated insulin secretion mediated by granule-localized K(ATP) channels by using conditions that bypass glibenclamide action on plasmalemmal K(ATP) channels. High-potassium stimulation evoked a sustained rise in beta-cell calcium level but a transient rise in insulin secretion. With continued high-potassium depolarization, addition of glibenclamide dramatically enhanced insulin secretion without affecting calcium. These findings support the hypothesis that glibenclamide, or an increased ATP/ADP ratio, stimulates insulin secretion in part by binding at granule-localized K(ATP) channels that functionally contribute to sustained second-phase insulin secretion.

Human insulin vesicle dynamics during pulsatile secretion.

In healthy individuals, plasma insulin levels oscillate in both fasting and fed states. Numerous studies of isolated pancreata and pancreatic islets support the hypothesis that insulin oscillations arise because the underlying rate of insulin secretion also oscillates; yet, insulin secretion has never been observed to oscillate in individual pancreatic beta-cells. Using expressed fluorescent vesicle cargo proteins and total internal reflection fluorescence (TIRF) microscopy, we demonstrate that glucose stimulates human pancreatic beta-cells to secrete insulin vesicles in short, coordinated bursts of approximately 70 vesicles each. Randomization tests and spectral analysis confirmed that the temporal patterns of secretion were not random, instead exhibiting alternating periods of secretion and rest, recurring with statistically significant periods of 15-45 s. Although fluorescent vesicles arrived at the plasma membrane before, during, and after stimulation, their rate of arrival was significantly slower than their rate of secretion, so that their density near the plasma membrane dropped significantly during the cell's response. To study in greater detail the vesicle dynamics during cyclical bursts of secretion, we applied trains of depolarizations once a minute and performed simultaneous membrane capacitance measurements and TIRF imaging. Surprisingly, young fluorescent insulin vesicles contributed at least half of the vesicles secreted in response to a first train, even though young vesicles were vastly outnumbered by older, nonfluorescent vesicles. For subsequent trains, young insulin vesicles contributed progressively less to total secretion, whereas capacitance measurements revealed that total stimulated secretion did not decrease. These results suggest that in human pancreatic beta-cells, young vesicles are secreted first, and only then are older vesicles recruited for secretion.

Carbon monoxide differentially inhibits TLR signaling pathways by regulating ROS-induced trafficking of TLRs to lipid rafts.

Carbon monoxide (CO), a byproduct of heme catabolism by heme oxygenase (HO), confers potent antiinflammatory effects. Here we demonstrate that CO derived from HO-1 inhibited Toll-like receptor (TLR) 2, 4, 5, and 9 signaling, but not TLR3-dependent signaling, in macrophages. Ligand-mediated receptor trafficking to lipid rafts represents an early event in signal initiation of immune cells. Trafficking of TLR4 to lipid rafts in response to LPS was reactive oxygen species (ROS) dependent because it was inhibited by diphenylene iodonium, an inhibitor of NADPH oxidase, and in gp91(phox)-deficient macrophages. CO selectively inhibited ligand-induced recruitment of TLR4 to lipid rafts, which was also associated with the inhibition of ligand-induced ROS production in macrophages. TLR3 did not translocate to lipid rafts by polyinosine-polycytidylic acid (poly(I:C)). CO had no effect on poly(I:C)-induced ROS production and TLR3 signaling. The inhibitory effect of CO on TLR-induced cytokine production was abolished in gp91(phox)-deficient macrophages, also indicating a role for NADPH oxidase. CO attenuated LPS-induced NADPH oxidase activity in vitro, potentially by binding to gp91(phox). Thus, CO negatively controlled TLR signaling pathways by inhibiting translocation of TLR to lipid rafts through suppression of NADPH oxidase-dependent ROS generation.

Derlin-1 promotes the efficient degradation of the cystic fibrosis transmembrane conductance regulator (CFTR) and CFTR folding mutants.

A complex involving Derlin-1 and p97 mediates the retrotranslocation and endoplasmic reticulum (ER)-associated degradation of misfolded proteins in yeast and is used by certain viruses to promote host cell protein degradation (Romisch, K. (2005) Annu. Rev. Cell Dev. Biol. 21, 435-456; Lilley, B. N., and Ploegh, H. L. (2004) Nature 429, 834-840; Ye, Y., Shibata, Y., Yun, C., Ron, D., and Rapoport, T. A. (2004) Nature 429, 841-847). We asked whether the components of this pathway are involved in the endoplasmic reticulum-associated degradation of the mammalian integral membrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR), a substrate for the ubiquitin-proteasome system. We report that Derlin-1 and p97 formed complexes with CFTR in human airway epithelial cells. Derlin-1 interacted with nonubiquitylated CFTR, whereas p97 associated with ubiquitylated CFTR. Exogenous expression of Derlin-1 led to its co-localization with CFTR in the ER where it reduced wild type (WT) CFTR expression and efficiently degraded the disease-associated CFTR folding mutants, DeltaF508 and G85E (>90%). Consistent with this, Derlin-1 also reduced the amount of WT or DeltaF508 CFTR appearing in detergent-in-soluble aggregates. An approximately 70% knockdown of endogenous Derlin-1 by RNA interference increased the steady-state levels of WT and DeltaF508 CFTR by 10-15-fold, reflecting its significant role in CFTR degradation. Derlin-1 mediated the degradation of N-terminal CFTR fragments corresponding to the first transmembrane domain of CFTR, but CFTR fragments that incorporated additional domains were degraded less efficiently. These findings suggest that Derlin-1 recognizes misfolded, nonubiquitylated CFTR to initiate its dislocation and degradation early in the course of CFTR biogenesis, perhaps by detecting structural instability within the first transmembrane domain.

Ligand-dependent linkage of the ATP site to inhibition gate closure in the KATP channel.

Major advances have been made on the inhibition gate and ATP site of the K(ir)6.2 subunit of the K(ATP) channel, but little is known about conformational coupling between the two. ATP site mutations dramatically disrupt ATP-dependent gating without effect on ligand-independent gating, observed as interconversions between active burst and inactive interburst conformations in the absence of ATP. This suggests that linkage between site and gate is conditionally dependent on ATP occupancy. We studied all substitutions at position 334 of the ATP site in K(ir)6.2deltaC26 that express in Xenopus oocytes. All substitutions disrupted ATP-dependent gating by 10-fold or more. Only positive-charged arginine or lysine at 334, however, slowed ligand-independent gating from the burst, and this was in some but not all patches. Moreover, the polycationic peptide protamine reversed the slowed gating from the burst of 334R mutant channels, and speeded the slow gating from the burst of wild-type SUR1/K(ir)6.2 in the absence of ATP. Our results support a two-step ligand-dependent linkage mechanism for K(ir)6.2 channels in which ATP-occupied sites function to electrostatically dissociate COOH-terminal domains from the membrane, then as in all K(ir) channels, free COOH-terminal domains and inner M2 helices transit to a lower energy state for gate closure.

Fluorescent cargo proteins in pancreatic beta-cells: design determines secretion kinetics at exocytosis.

We compared secretion kinetics for four different fluorescent cargo proteins, each targeted to the lumen of insulin secretory vesicles. Upon stimulation, individual vesicles displayed one of four distinct patterns of fluorescence change: i), disappearance, ii), dimming, iii), transient brightening, or iv), persistent brightening. For each fusion protein, a different pattern of fluorescence change dominated. Furthermore, we demonstrated that the dominant pattern depends upon both i), the specific choice of fluorescent protein, and ii), the sequence of amino acids linking the cargo protein to the fluorescent protein. Thus, in beta-cells, experiments involving fluorescent cargo proteins for the study of exocytosis must be interpreted carefully, as design of a fluorescent cargo protein determines secretion kinetics at exocytosis.

Concerted gating mechanism underlying KATP channel inhibition by ATP.

KATP channels assemble from four regulatory SUR1 and four pore-forming Kir6.2 subunits. At the single-channel current level, ATP-dependent gating transitions between the active burst and the inactive interburst conformations underlie inhibition of the KATP channel by intracellular ATP. Previously, we identified a slow gating mutation, T171A in the Kir6.2 subunit, which dramatically reduces rates of burst to interburst transitions in Kir6.2DeltaC26 channels without SUR1 in the absence of ATP. Here, we constructed all possible mutations at position 171 in Kir6.2DeltaC26 channels without SUR1. Only four substitutions, 171A, 171F, 171H, and 171S, gave rise to functional channels, each increasing Ki,ATP for ATP inhibition by >55-fold and slowing gating to the interburst by >35-fold. Moreover, we investigated the role of individual Kir6.2 subunits in the gating by comparing burst to interburst transition rates of channels constructed from different combinations of slow 171A and fast T171 "wild-type" subunits. The relationship between gating transition rate and number of slow subunits is exponential, which excludes independent gating models where any one subunit is sufficient for inhibition gating. Rather, our results support mechanisms where four ATP sites independently can control a single gate formed by the concerted action of all four Kir6.2 subunit inner helices of the KATP channel.

Body window-enabled in vivo multicolor imaging of transplanted mouse islets expressing an insulin-Timer fusion protein.

Type 1 diabetes results from the selective destruction of insulin-producing beta cells in the islets of Langerhans, and autoimmune T cells are thought to be the mediators of this destruction. T cells are also responsible for allorejection once the islets are transplanted into a patient to reduce the negative consequences of a lack of insulin. To better understand these processes, we have developed a transgenic mouse expressing proinsulin II tagged with a live-cell fluorescent reporter protein, Timer. Timer protein is unique because it changes color from green to red in the first 24 h after synthesis. With this marker, insulin synthesis can be carefully monitored through fluorescent changes over time. To complement this new biotechnological research tool, we designed a body window to allow for in vivo imaging over time of the islets transplanted under the kidney capsule. The window device, which is sutured to replace the underlying skin and body wall over the site of islet transplantation, may be used to simultaneously observe beta cells and T cells that have been labeled with a fluorochrome distinguishable from Timer. The imaging of both insulin-producing cells and T cells may be carried out repeatedly for a week or more with no need for repeated surgery, while preserving the life of the studied animal.

The insulin secretory granule is the major site of K(ATP) channels of the endocrine pancreas.

With ATP sites on K(ir)6.2 that inhibit activity and ADP sites on SUR1 that antagonize the inhibition, ATP-sensitive potassium channels (K(ATP) channels) are designed as exquisite sensors of adenine nucleotide levels that signal changes in glucose metabolism. If pancreatic K(ATP) channels localize to the insulin secretory granule, they would be well positioned to transduce changes in glucose metabolism into changes in granule transport and exocytosis. Tests for pancreatic K(ATP) channels localized to insulin secretory granules led to the following observations: fluorescent sulfonylureas that bind the pancreatic K(ATP) channel specifically label intracellular punctate structures in cells of the endocrine pancreas. The fluorescent glibenclamides colocalize with Ins-C-GFP, a live-cell fluorescent reporter of insulin granules. Expression of either SUR1-GFP or K(ir)6.2-GFP fusion proteins, but not expression of GFP alone, directs GFP fluorescence to insulin secretory granules. An SUR1 antibody specifically labels insulin granules identified by anti-insulin. Two different K(ir)6.2 antibodies specifically label insulin secretory granules identified by anti-insulin. Immunoelectron microscopy showed K(ir)6.2 antibodies specifically label perimeter membrane regions of the secretory granule. Relatively little or no labeling of other structures, including the plasma membrane, was found. Our results demonstrate that the insulin secretory granule is the major site of K(ATP) channels of the endocrine pancreas.

A functional link between glucokinase binding to insulin granules and conformational alterations in response to glucose and insulin.

Glucokinase (GK) activity is essential for the physiological regulation of insulin secretion by glucose. Because the enzyme exerts nearly total control over glucose metabolism in the beta-cell, even small changes in GK activity exert effects on glucose-stimulated insulin secretion and, consequently, the blood glucose concentration. Using quantitative imaging of multicolor fluorescent proteins fused to GK, we found that the association of GK with insulin granules is regulated by glucose in the beta-cell. Glucose stimulation increased the rate of fluorescence recovery after photobleaching of GK to insulin granules, indicating that GK is released into the cytoplasm after glucose stimulation. Changes in fluorescence resonance energy transfer between two different fluorescent protein variants inserted on opposing ends of GK were observed after glucose stimulation and correlated with increased enzyme activity. Furthermore, glucose-stimulated changes in GK regulation were blocked by two inhibitors of insulin secretion. Insulin treatment restored GK regulation in inhibited cells and stimulated GK translocation and activation by itself. Together, these data support a model for post-translational regulation of GK whereby insulin regulates both the association of GK with secretory granules and the activity of the enzyme within the pancreatic beta-cell.