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 β-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 β-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 β-cell. 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 β-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 β-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 β-cell. glucokinase glucose-stimulated insulin secretion fluorescence resonance energy transfer fluorescence recovery after photobleaching green fluorescent protein cyan fluorescent protein yellow fluorescent protein hexokinase maturity onset diabetes of the young, type 2 phosphate-buffered saline Glucokinase (GK)1 plays an essential role in glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells by determining the rate of metabolic flux in response to changes in the plasma glucose concentration (1Matschinsky F.M. Glaser B. Magnuson M.A. Diabetes. 1998; 47: 307-315Crossref PubMed Scopus (288) Google Scholar). Small changes in GK activity have large effects on the rate of GSIS, as indicated by studies of both humans with maturity onset diabetes of the young, type 2 (MODY2) (2Wang H. Iynedjian P.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4372-4377Crossref PubMed Scopus (115) Google Scholar, 3Glaser B. Kesavan P. Heyman M. Davis E. Cuesta A. Buchs A. Stanley C.A. Thornton P.S. Permutt M.A. Matschinsky F.M. Herold K.C. N. Engl. J. Med. 1998; 338: 226-230Crossref PubMed Scopus (508) Google Scholar, 4Davis E.A. Cuesta-Munoz A. Raoul M. Buettger C. Sweet I. Moates M. Magnuson M.A. Matschinsky F.M. Diabetologia. 1999; 42: 1175-1186Crossref PubMed Scopus (116) Google Scholar), and tissue-specific gene knock-out mice (5Postic C. Shiota M. Niswender K.D. Jetton T.L. Chen Y. Moates J.M. Shelton K.D. Lindner J. Cherrington A.D. Magnuson M.A. J. Biol. Chem. 1999; 274: 305-315Abstract Full Text Full Text PDF PubMed Scopus (1004) Google Scholar). It has long been known that glucose is an important regulator of GK activity in the β-cell, although the mechanisms through which glucose acts to regulate GK activity in the β-cell are not yet understood. Several studies have suggested that glucose influences GK activity by affecting either the rate of gene transcription or by altering the stability of the enzyme itself (1Matschinsky F.M. Glaser B. Magnuson M.A. Diabetes. 1998; 47: 307-315Crossref PubMed Scopus (288) Google Scholar, 6Liang Y. Najafi H. Matschinsky F.M. J. Biol. Chem. 1990; 265: 16863-16866Abstract Full Text PDF PubMed Google Scholar, 7Gasa R. Fabregat M.E. Gomis R. Biochem. Biophys. Res. Commun. 2000; 268: 491-495Crossref PubMed Scopus (13) Google Scholar). However, these studies were not designed to explore the regulation of GK at the post-translational level, as was suggested by several studies that reported glucose-sensitive changes in GK activity independent of changes in GK protein levels (8Liang Y. Najafi H. Smith R.M. Zimmerman E.C. Magnuson M.A. Tal M. Matschinsky F.M. Diabetes. 1992; 41: 792-806Crossref PubMed Scopus (147) Google Scholar, 9Chen C. Bumbalo L. Leahy J.L. Diabetes. 1994; 43: 684-689Crossref PubMed Scopus (35) Google Scholar, 10Chen C. Hosokawa H. Bumbalo L.M. Leahy J. J. Clin. Invest. 1994; 94: 1616-1620Crossref PubMed Scopus (56) Google Scholar). Thus, post-translational regulation of GK in the β-cell has not been clearly established, nor has a feasible mechanistic explanation for these data emerged. One potential avenue for post-translational modulation of GK function has arisen from recent observations that GK is associated with insulin secretory granules (11Toyoda Y. Yoshie S. Shironoguchi H. Miwa I. Histochem. Cell Biol. 1999; 112: 35-40Crossref PubMed Scopus (19) Google Scholar, 12Stubbs M. Aiston S. Agius L. Diabetes. 2000; 49: 2048-2055Crossref PubMed Scopus (27) Google Scholar). Because agonist-stimulated changes in the cellular localization of proteins is an important mechanism for regulating protein function (13Teruel M.N. Meyer T. Cell. 2000; 103: 181-184Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar), and because regulation of GK in the liver is known to involve flux between the cytoplasm and the nucleus (14Agius L. Peak M. Biochem. J. 1993; 296: 785-796Crossref PubMed Scopus (131) Google Scholar, 15Shiota C. Coffey J. Grimsby J. Grippo J.F. Magnuson M.A. J. Biol. Chem. 1999; 274: 37125-37130Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar), the prospect of GK regulation in the β-cell involving reversible association with secretory granules presented an attractive hypothesis. GK association with a variety of bound and cytoplasmic compartments has been described in recent years (12Stubbs M. Aiston S. Agius L. Diabetes. 2000; 49: 2048-2055Crossref PubMed Scopus (27) Google Scholar, 16Noma Y. Bonner-Weir S. Latimer J.B. Davalli A.M. Weir G.C. Endocrinology. 1996; 137: 1485-1491Crossref PubMed Scopus (48) Google Scholar, 17Vanhoutte C. Malaisse W.J. Mol. Genet. Metab. 1998; 63: 176-182Crossref PubMed Scopus (5) Google Scholar, 18Tiedge M. Steffeck H. Elsner M. Lenzen S. Diabetes. 1999; 48: 514-523Crossref PubMed Scopus (42) Google Scholar); however, these studies have not established a clear role for different GK-associated compartments in the response of the β-cell to glucose. Indeed, it is not known whether translocation between compartments even occurs. Thus, although evidence exists for the multicompartment localization of GK in β-cells, the significance of this compartmentalization, the mechanisms involved, and functional impact of alterations in GK localization remain controversial, if not wholly uncertain. One potential explanation for the lack of consensus in examining GK localization arises from the low resolution methods employed in these studies, namely biochemical fractionation techniques and immunofluorescent microscopy. Low resolution techniques, such as immunolabeling (12Stubbs M. Aiston S. Agius L. Diabetes. 2000; 49: 2048-2055Crossref PubMed Scopus (27) Google Scholar) and biochemical fractionation (18Tiedge M. Steffeck H. Elsner M. Lenzen S. Diabetes. 1999; 48: 514-523Crossref PubMed Scopus (42) Google Scholar), are of limited value in studying the association of GK with insulin granules because these techniques require destruction of the sample prior to analysis. It is especially difficult to apply these techniques to the study of proteins that are expressed in low levels, as is GK expression in β-cells, because even small losses in protein have a large impact on efforts to quantify changes in protein localization. Furthermore, these methods provide little temporal information, making characterization of dynamic processes, such as GK translocation, exceedingly difficult. Therefore, the study of GK localization in β-cells would greatly benefit from an alternative approach. Recent developments in fluorescent protein-based techniques, along with improvements in live cell imaging technology, now enable measurements of a wide variety of biochemical processes in living cells. One such technique that has been successfully used to monitor changes in protein localization and mobility in living cells is fluorescence recovery after photobleaching (FRAP) (19Cole N.B. Smith C.L. Sciaky N. Terasaki M. Edidin M. Lippincott-Schwartz J. Science. 1996; 273: 797-801Crossref PubMed Scopus (400) Google Scholar, 20Henkel A.W. Simpson L.L. Ridge R.M. Betz W.J. J. Neurosci. 1996; 16: 3960-3967Crossref PubMed Google Scholar, 21Vasudevan C. Han W. Tan Y. Nie Y., Li, D. Shome K. Watkins S.C. Levitan E.S. Romero G. J. Cell Sci. 1998; 111: 1277-1285Crossref PubMed Google Scholar, 22Reits E.A.J. Neefjes J.J. Nat. Cell Biol. 2001; 3: E145-E147Crossref PubMed Scopus (500) Google Scholar). In FRAP, fluorophores in a region of the cell are irreversibly photobleached, which markedly reduces the signal in that region. As photobleached fluorophores diffuse away from the region, they are replaced by unbleached fluorophores that diffuse in from outside the photobleached region. Thus, the fluorescence signal recovers with time. If the photobleached fluorophores are bound to stationary intracellular components, then the fluorescence signal will not recover. Therefore, FRAP can be used to measure not only diffusion, but also binding sites within the cell. Another technique, fluorescence resonance energy transfer (FRET), has been used to expand the spatial resolution limit to the <100-Å range, thereby permitting detection of changes in protein conformation and protein-protein interactions (23Patterson G.H. Piston D.W. Barisas B.G. Anal. Biochem. 2000; 284: 438-440Crossref PubMed Scopus (314) Google Scholar, 24Pollok B.A. Heim R. Trends Cell Biol. 1999; 9: 57-60Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar, 25van Roessel P. Brand A.H. Nat. Cell Biol. 2002; 4: E15-E20Crossref PubMed Scopus (208) Google Scholar, 26Janetopoulos C. Jin T. Devreotes P. Science. 2001; 291: 2408-2411Crossref PubMed Scopus (374) Google Scholar). These two techniques allow effective quantification of subtle changes in protein localization and structure that are often masked by the signal to noise limitations, finite collection ranges, and the physical resolution limits of current microscope systems. Not surprisingly, these high resolution techniques, when used in conjunction with more traditional approaches, have led to major advances in our understanding of protein motion and localization in the context of their natural cellular environment. Here, we have utilized FRAP, FRET, and biochemical fractionation to explore post-transcriptional regulation of GK in the β-cell. We report that, in the absence of glucose, GK is associated with insulin granules, as determined by FRAP assays and biochemical fractionation. Glucose leads to a release of GK from this bound state into the cytoplasm. Furthermore, the release of GK from the granule-bound state is accompanied both by increased GK activity and a conformational change, as detected using FRET. We also found that either the inhibition of insulin secretion or insulin receptor function blocked glucose-stimulated GK translocation and conformational changes. Moreover, insulin was able to stimulate GK translocation and activation in the absence of glucose. Together, these results point to the regulation of GK localization and activity by insulin secreted from β-cells in response to glucose. These data provide an important advance to our understanding of GSIS from pancreatic β-cells and may have important implications in the pathogenesis and treatment of diabetes. Fluorescent protein expression vectors were obtained from BD CLONTECH. Fluorescently tagged secondary antibodies were obtained from Molecular Probes. Chemicals were from Sigma-Aldrich unless otherwise noted. Restriction enzymes were obtained from New England Biolabs. PCR primers were purchased from Integrated DNA Technologies. DNA isolation reagents were obtained from Qiagen. Optical filters were obtained from Chroma. Peroxidase conjugated secondary antibodies were obtained from Jackson Immunoresearch. Cell culture reagents were obtained from Media and Reagents Core of the Diabetes Research and Training Center at Vanderbilt University. A cDNA encoding the rat β-cell GK isoform was inserted into the pEGFP-N3 vector. A pEYFP-N3 vector was made by inserting YFP from the pEYFP-N1 vector into the same position as the GFP in the N3 vector. β-Cell GK cDNA was then inserted into this construct to make GK-YFP. CFP-GK-YFP was made by transfer of CFP sequences from pECFP-N1 into the GK-YFP plasmid. The C-peptide CFP construct was made by inserting CFP into the murine proinsulin II cDNA at the native XmaI site in the C-peptide region of the gene. This construct was generated in two parts by splicing the N-terminal portion of the gene into pECFP-N3 (generated as described above for the pEYFP-N3 vector) and inserting the C-terminal portion into the pECFP-C1 construct. The full-length construct was made by insertion of the BsrGI,XbaI fragment of the C-terminal portion into the vector containing the N-terminal fragment. The dsRed C-peptide construct was made by removal of the fluorescent protein from the C-peptide CFP construct and insertion of dsRed cDNA. Details of the assembly and the plasmid maps are available upon request. All plasmid constructs were verified by sequencing reactions performed by the Vanderbilt-Ingram Cancer Center DNA Sequencing Shared Resource. Plasmid DNAs were introduced into βTC3 cells by 10 50-μs square wave pulses of 300 V at 500-ms intervals with a BTX ECM830 electroporator. 5 μg of each construct was used per 20% of a 80% confluent T-75 flask of βTC3 cells suspended in Dulbecco's PBS (BioWhittaker) in a 2-mm gapped cuvette. Cells were grown in Dulbecco's modified Eagle's medium containing 15% horse serum and 2.5% fetal bovine serum with high glucose and switched to 5 mm glucose medium 24 h prior to experimentation. Cells were starved for 2–3 h in glucose-free BMHH plus 0.1% bovine serum albumin (27Piston D.W. Knobel S.M. Postic C. Shelton K.D. Magnuson M.A. J. Biol. Chem. 1999; 274: 1000-1004Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) prior to experimentation. For immunofluorescent staining, cells were grown on coverslips and fixed for 30 min with fresh 4% paraformaldehyde in PBS at 4 °C. Cells were permeabilized with 0.1% Triton X-100, blocked with 3% bovine serum albumin, and stained with rabbit anti-GK (28Jetton T.L. Magnuson M.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2619-2623Crossref PubMed Scopus (84) Google Scholar) and/or guinea pig anti-insulin (Linco) where indicated. Anti-rabbit ALEXA-488 and anti-guinea pig ALEXA-546 were applied for the detection of GK and insulin immunostains where appropriate. Visualization was with a Zeiss LSM510 microscope (63×, 1.4 numeric aperture apochromat) using the 457-, 488-, and 514-nm argon lines and a 543-nm helium-neon laser to excite CFP, GFP/ALEXA-488, YFP, and dsRed/ALEXA-546, respectively. Emitted light was passed through bandpass filters for collection of CFP (470–510 nm), GFP/Alexa-488 (505–530 nm), and YFP (530–550 nm). Long-pass filters were used for collection of Alexa-546 (560 nm) and dsRed (590 nm). Examination of dsRed was in cells expressing this construct for at least 48 h, when emission in the "green state" of the fluorophore was no longer detectable using our standardized optical conditions. FRAP measurements were taken using procedures similar to those that have been reported as biologically useful without prohibitory phototoxic effects (19Cole N.B. Smith C.L. Sciaky N. Terasaki M. Edidin M. Lippincott-Schwartz J. Science. 1996; 273: 797-801Crossref PubMed Scopus (400) Google Scholar, 20Henkel A.W. Simpson L.L. Ridge R.M. Betz W.J. J. Neurosci. 1996; 16: 3960-3967Crossref PubMed Google Scholar, 21Vasudevan C. Han W. Tan Y. Nie Y., Li, D. Shome K. Watkins S.C. Levitan E.S. Romero G. J. Cell Sci. 1998; 111: 1277-1285Crossref PubMed Google Scholar, 22Reits E.A.J. Neefjes J.J. Nat. Cell Biol. 2001; 3: E145-E147Crossref PubMed Scopus (500) Google Scholar), such as cell death. In our experiments, we observed no morphological changes in the cells, as would be expected from cellular photodamage. Quantification of the FRET ratio of granules was performed by selecting regions that were positive for dsRed fluorescence and calculating the ratio of YFP/CFP intensities in these regions using NIH Image software. Regions that contained no dsRed fluorescence were determined to be cytoplasmic areas. Prestimulation ratios from whole cells were normalized to 1.0 for comparison purposes in Fig. 4. Living cells were maintained at 32 °C using the Bioptechs Delta T open dished system. Identical results were obtained at 37 °C, although maintaining focus on single granules became more problematic because of increased granule motion and focal drift. Fluorescence spectra of CFP-GK-YFP was obtained from cell lysates (1.5% Triton X-100, PBS, 30 min) using a spectrofluorimeter. Spectra were normalized to cells not expressing the construct. Following stimulation as described, cells were collected in cold PBS and resuspended in cold 50 mm HEPES buffer (pH 7.2) containing 125 mm KCl, 20 mm NaCl, 0.5 mm CaCl2, 0.5 mm MgCl2, along with 20 μg/ml digitonin. Cells were incubated on ice for 10 min prior to centrifugation in a microcentrifuge (20 min at 12,000 × g). Pellet and supernatant fractions were then analyzed by SDS-PAGE electrophoresis and Western blot using rabbit anti-GK antibodies and peroxidase-conjugated secondary antibodies. Observation of GK was by film exposure to nitrocellulose membranes treated with ECL+PLUS Western blot Detection System (Amersham Biosciences). GK activity from an equal number of cells lysed with 1.5% Triton X-100 was assayed by a glucose-6-phosphate dehydrogenase-coupled reaction according to the protocol of Fernandez-Mejia et al. (29Fernandez-Mejia C. Vega-Allende J. Rojas-Ochoa A. Rodriguez-Dorantes M. Romero-Navarro G. Matschinsky F.M. Wang J. German M.S. Endocrinology. 2001; 142: 1448-1452Crossref PubMed Scopus (20) Google Scholar) using NAD as a coenzyme. NADH production was quantified using a spectrofluorimeter (350-nm excitation, 450-nm collection). Single measurements were generated from triplicate measurements of single samples. In addition, the protein concentration of the cell lysates was determined using the Advanced Protein Assay Reagent (Cytoskeleton, Inc.) atA590 to ensure proper normalization across samples. Total cellular GK activity was measured by subtraction of the activity measured at 0.5 mm glucose from the activity measured at 100 mm glucose to account for hexokinase activity in our lysates. Colocalization between GK and insulin granules in βTC3 cells was detected using an affinity-purified anti-GK antibody (28Jetton T.L. Magnuson M.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2619-2623Crossref PubMed Scopus (84) Google Scholar) in an immunofluorescent staining experiment (Fig.1A). This finding agrees with previous investigations into the distribution of GK in primary β-cells and insulinoma cell lines (11Toyoda Y. Yoshie S. Shironoguchi H. Miwa I. Histochem. Cell Biol. 1999; 112: 35-40Crossref PubMed Scopus (19) Google Scholar, 12Stubbs M. Aiston S. Agius L. Diabetes. 2000; 49: 2048-2055Crossref PubMed Scopus (27) Google Scholar). In addition, similar results were obtained using MIN6 cells and INS1 cells (data not shown), thereby supporting the notion that the association of GK with insulin granules is a phenomena that is not limited to certain cell lines. To more directly assess the interaction of GK with insulin granules, GK-YFP was made and expressed in cells along with a CFP construct that was targeted to insulin granules by insertion of CFP into the connecting peptide segment of the murine proinsulin II cDNA (C-peptide CFP) (30Watkins S. Geng X., Li, L. Papworth G. Robbins P.D. Drain P. Traffic. 2002; 3: 461-471Crossref PubMed Scopus (39) Google Scholar). As shown in Fig. 1B, the distribution of these constructs was identical to the distribution of endogenous GK and insulin granules, indicating that this system was appropriate for monitoring the association of GK with insulin granules in living cells. Moreover, a GFP-tagged hexokinase II (HK-GFP) variant (15Shiota C. Coffey J. Grimsby J. Grippo J.F. Magnuson M.A. J. Biol. Chem. 1999; 274: 37125-37130Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar) did not localize to granules (Fig. 1C). This suggests that granule association occurs only with GK and is not a general property of all hexokinase isoforms. Quantification of the association between GK and insulin granules is a complex problem because the "bound" and "free" compartments are not readily distinguished from each other. Indeed, the bound compartment in this case involves granules that are, themselves, mobile within the free cytoplasmic compartment, thus making it difficult to measure changes in GK distribution. To be able to quantify changes in GK distribution in β-cells, we used the highly sensitive FRAP technique to measure the release of GK-YFP from CFP-labeled insulin granules into the cytoplasmic compartment. As shown in Fig.2A, a region containing several CFP-labeled granules was photobleached with a high intensity pulse of 514-nm laser to destroy the fluorescence from GK-YFP (31Patterson G. Day R.N. Piston D. J. Cell Sci. 2001; 114: 837-838Crossref PubMed Google Scholar) and the recovery of GK-YFP to the CFP-labeled granules was measured. The analysis was performed only on granules that remained in place during the course of the experiment, as indicated by the CFP fluorescence. Recovery of the GK-YFP to the photobleached granules occurred over several seconds and was considerably slower than recovery in the cytoplasm (Fig. 2A, red dotted line), which recovers with a t12 of ∼100 ms (32Luby-Phelps K. Taylor D.L. Lanni F. J. Cell Biol. 1986; 102: 2015-2022Crossref PubMed Scopus (246) Google Scholar). Although GK-YFP fluorescence recovered to granules after stimulation with at least 1 mm glucose for 30 min, it did not recover to granules in cells cultured with less than 1 mm glucose (Fig. 2B). At high concentrations of glucose (>10 mm), it is expected that the total amount of GK-YFP associated with the granules would decrease. However, recovery of GK-YFP to granules at higher concentrations was still much slower that recovery to the cytoplasm (Fig. 2A), indicating that recovery at these concentrations cannot be explained by simple diffusion of unbound GK into the field. Translocation of the GK-YFP was also examined using a digitonin permeabilization assay (Fig. 2C). In this case, βTC3 cells were incubated with increasing glucose concentrations prior to treatment with digitonin and separation of the cell into both soluble (cytoplasmic) and membrane-bound (pellet) compartments by centrifugation. Under low glucose conditions, GK was detected only in the membrane-bound fraction. Increasing glucose concentrations resulted in a gradual redistribution of GK to the soluble compartment, corresponding with the dose-response data obtained from FRAP measurements. Together, these observations indicate that GK colocalizes with insulin granules in β-cells and that the enzyme translocates from the bound to the free compartment in response to changes in the glucose concentration. Based on these observations, we hypothesized that GK translocation in the β-cell may constitute a potential mechanism for acute regulation of GK activity. To test this possibility, we measured GK activity under the conditions that resulted either in the tight association of GK with granules (glucose starvation) or reduced granule binding and release to the cytoplasm (10 mm glucose). Stimulation of cells with 10 mm glucose for 1 h resulted in increasing the cellular GK activity 40% when compared with untreated cells (Fig.3C) (29Fernandez-Mejia C. Vega-Allende J. Rojas-Ochoa A. Rodriguez-Dorantes M. Romero-Navarro G. Matschinsky F.M. Wang J. German M.S. Endocrinology. 2001; 142: 1448-1452Crossref PubMed Scopus (20) Google Scholar) (p < 0.05, by t test) when compared with unstimulated cells. This increase in GK activity is consistent with cytoplasmic GK having a higher activity than granule-bound GK. However, the GK activity assay was not sensitive enough to examine the relative activity of the bound and free compartments, although increased GK activity occurs under conditions where translocation to the cytoplasm has been observed. Therefore, we developed a fluorescence-based probe for examining different GK conformational and/or activity states. Changes in protein activity often reflect alterations in their conformational state and such conformational changes can be directly measured by examination of FRET between fluorescent proteins of different colors (23Patterson G.H. Piston D.W. Barisas B.G. Anal. Biochem. 2000; 284: 438-440Crossref PubMed Scopus (314) Google Scholar). To make use of this fact, we assembled a GK fusion protein that was tagged on both the C- and N-terminal ends by attaching CFP as a donor chromophore to the N terminus of GK-YFP. Analysis of the emission spectra of extracts from cells expressing CFP-GK-YFP revealed that glucose stimulation was accompanied by a shift in the emission spectra (Fig. 3A). The relative FRET ratio of this probe was calculated from samples treated identically to those where GK activation was observed (Fig. 3, B andC), suggesting that the changes to the FRET signal from the CFP-GK-YFP construct reports changes in GK conformation that correlate with increased activity (Fig. 3D). To assess the changes in FRET spectra in single living cells, the CFP-GK-YFP construct was introduced into cells along with a C-peptide construct containing the dsRed fluorescent protein. As shown in Fig. 3 (E andF), granule-bound CFP-GK-YFP displayed a strong FRET signal, whereas cytoplasmic CFP-GK-YFP displayed a weak FRET signal. These results are consistent with GK having a lower activity when associated with insulin granules and a higher activity when released into the cytoplasm Because the quantitative imaging data we obtained supports the post-translational regulation of GK by glucose, we next focused on a mechanism that would satisfactorily explain the effect of glucose on GK activity and localization. The simplest potential mechanism for glucose-induced regulation of GK would be the induction of GK translocation and activation upon the binding of glucose to GK. Because glucose enters β-cells through facilitated diffusion, this hypothesis predicts that regulation of GK would occur concurrent with the addition of the glucose bolus. However, we did not find evidence of changes in GK localization (as determined by our FRAP assay) until 20 min after stimulation with glucose (Fig.4A). Because regulation of GK occurs much later than glucose stimulation, it is not adequately explained by simple association between GK and glucose. Moreover, the glucose concentration-response profile of this process (see above) was not consistent with reported affinities for glucose-GK interactions (Km ∼ 8 mm (Ref. 1Matschinsky F.M. Glaser B. Magnuson M.A. Diabetes. 1998; 47: 307-315Crossref PubMed Scopus (288) Google Scholar)). Recent studies have shown a role for insulin signaling in β-cells in maintaining normal GSIS (33Kulkarni R.N. Bruning J.C. Winnay J.N. Postic C. Magnuson M.A. Kahn C.R. Cell. 1999; 96: 329-339Abstract Full Text Full Text PDF PubMed Scopus (929) Google Scholar, 34Aspinwall C.A. Qian W.J. Roper M.G. Kulkarni R.N. Kahn C.R. Kennedy R.T. J. Biol. Chem. 2000; 275: 22331-22338Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Indeed, islets from mice that lack insulin receptors exhibit impaired GSIS despite maintaining a normal response to arginine (33Kulkarni R.N. Bruning J.C. Winnay J.N. Postic C. Magnuson M.A. Kahn C.R. Cell. 1999; 96: 329-339Abstract Full Text Full Text PDF PubMed Scopus (929) Google Scholar). Furthermore, insulin receptor signaling stimulates a broad range of intracellular signaling pathways in the β-cell, which modulate a wide variety of cellular functions. Because evidence for the regulation of GSIS by an insulin autocrine/paracrine feedback loop has recently emerged, we performed experiments to test the hypothesis that GSIS and the subsequent autostimulation of insulin receptors on the β-cell is the signal that leads to both GK translocation and activation. To assess the importance of insulin signaling in glucose-stimulated GK regulation, we pretrea