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• W1965867110 abstract "We have shown that intermediate lobe (IL) pituitary cells can be engineered to produce sufficient amounts of insulin (ins) to cure diabetes in nonobese diabetic mice but, unlike transplanted islets, ILins cells evade immune attack. To confer glucose-sensing capabilities into these cells, they were further modified with recombinant adenoviruses to express high levels of GLUT2 and the β-cell isoform of glucokinase (GK). Although expression of GLUT2 alone had negligible effects on glucose usage and lactate production, expression of GK alone resulted in ∼2-fold increase in glycolytic flux within the physiological (3–20 mm) glucose range. GLUT2/GK coexpression further increased glycolytic flux at 20 mm glucose but disproportionately increased flux at 3 mm glucose. Despite enhanced glycolytic fluxes, GLUT2/GK-coexpressing cells showed glucose dose-dependent accumulation of hexose phosphates, depletion of intracellular ATP, and severe apoptotic cell death. These studies demonstrate that glucose-sensing properties can be introduced into non-islet cells by the single expression of GK and that glucose responsiveness can be augmented by the coexpression of GLUT2. However, in the metabolic engineering of surrogate β cells, it is critical that the levels of the components be closely optimized to ensure their physiological function and to avoid the deleterious consequences of glucose-induced toxicity. We have shown that intermediate lobe (IL) pituitary cells can be engineered to produce sufficient amounts of insulin (ins) to cure diabetes in nonobese diabetic mice but, unlike transplanted islets, ILins cells evade immune attack. To confer glucose-sensing capabilities into these cells, they were further modified with recombinant adenoviruses to express high levels of GLUT2 and the β-cell isoform of glucokinase (GK). Although expression of GLUT2 alone had negligible effects on glucose usage and lactate production, expression of GK alone resulted in ∼2-fold increase in glycolytic flux within the physiological (3–20 mm) glucose range. GLUT2/GK coexpression further increased glycolytic flux at 20 mm glucose but disproportionately increased flux at 3 mm glucose. Despite enhanced glycolytic fluxes, GLUT2/GK-coexpressing cells showed glucose dose-dependent accumulation of hexose phosphates, depletion of intracellular ATP, and severe apoptotic cell death. These studies demonstrate that glucose-sensing properties can be introduced into non-islet cells by the single expression of GK and that glucose responsiveness can be augmented by the coexpression of GLUT2. However, in the metabolic engineering of surrogate β cells, it is critical that the levels of the components be closely optimized to ensure their physiological function and to avoid the deleterious consequences of glucose-induced toxicity. nonobese diabetic pro-opiomelanocortin intermediate lobe insulin insulin-producing intermediate lobe glucose transporter isotype 2 glucokinase ATP-sensitive potassium channels glucose-stimulated insulin secretion acetylcholine hexokinase phosphofructokinase glucose 6-phosphate glyceraldehyde 3-phosphate 6BP, fructose 1,6-bisphosphate adenovirus green fluorescence protein glyceraldehyde-3-phosphate dehydrogenase kilobase(s) multiplicity of infection terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling tetramethylrhodamine base pair(s) anterior lobe human growth hormone cytomegalovirus Type 1 diabetes in humans and in nonobese diabetic (NOD)1 mice is an autoimmune disorder that results in the complete destruction of the insulin-producing pancreatic β cells. Despite recent advances in insulin injection therapy, current regimens do not adequately mimic the normal physiological patterns of insulin release by β cells, and patients with diabetes are thus at continued risk for developing severe long-term complications. These limitations have provided the impetus to develop alternative forms of therapy that would circumvent the need for insulin injections.One appealing therapeutic strategy is to engineer insulin expression into non-β cells (1Lipes M.A. Cooper E.M. Skelly R. Rhodes C.J. Boschetti E. Weir G.C. Davalli A.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8595-8600Crossref PubMed Scopus (78) Google Scholar, 2Chen R. Meseck M. McEvoy R.C. Woo S.L. Gene Ther. 2000; 7: 1802-1809Crossref PubMed Scopus (56) Google Scholar, 3Cheung A.T. Dayanandan B. Lewis J.T. Korbutt G.S. Rajotte R.V. Bryer-Ash M. Boylan M.O. Wolfe M.M. Keiffer T.J. Science. 2000; 290: 1959-1962Crossref PubMed Scopus (256) Google Scholar). Using transgenic mouse techniques, we targeted mouse preproinsulin 2 (ins) gene expression to the intermediate lobe (IL) of the pituitary in NOD mice via the proopiomelanocortin (POMC) promoter (1Lipes M.A. Cooper E.M. Skelly R. Rhodes C.J. Boschetti E. Weir G.C. Davalli A.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8595-8600Crossref PubMed Scopus (78) Google Scholar). Our studies showed that the IL cells secreted large amounts of insulin, sufficient to cure diabetes when implanted into spontaneously diabetic NOD mice. Moreover, in contrast to transplanted islets, the insulin-producing IL (hereafter, ILins) cells evaded immune recognition and damage. These studies were the first to show that a non-islet cell type could be engineered to secrete sufficient amounts of insulin to cure diabetes, yet escape the autoimmune process that kills β cells in type 1 diabetes (1Lipes M.A. Cooper E.M. Skelly R. Rhodes C.J. Boschetti E. Weir G.C. Davalli A.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8595-8600Crossref PubMed Scopus (78) Google Scholar). However, a limitation of this insulin gene delivery system was that insulin secretion was not glucose-regulated.The regulation of insulin release in β cells is mediated by the metabolism of glucose (4Meglasson M.D. Matschinsky F.M. Diabetes Metab. Rev. 1986; 2: 163-214Crossref PubMed Scopus (412) Google Scholar). According to current models, glucose metabolism generates signals such as ATP or an increase in the ratio of ATP to ADP ratio that leads to closure of the ATP-sensitive K+ (KATP) channels. The resulting plasma membrane depolarization activates voltage-gated L-type Ca2+channels, inducing the influx of Ca2 that triggers insulin granule exocytosis (5Newgard C.B. McGarry J.D. Annu. Rev. Biochem. 1995; 64: 689-719Crossref PubMed Scopus (491) Google Scholar). The proteins that control the metabolic flux of glucose in β cells function as “glucose sensors.” Rodent β cells express two specialized proteins that have been considered as candidates for the glucose sensor: the facilitated glucose transporter isoform, GLUT2, and the low affinity glucose phosphorylating enzyme, glucokinase (GK). A large body of evidence suggests that GK is the flux-controlling enzyme for glycolysis in β cells and, as such, serves as the “gatekeeper” for metabolic signaling (4Meglasson M.D. Matschinsky F.M. Diabetes Metab. Rev. 1986; 2: 163-214Crossref PubMed Scopus (412) Google Scholar). GK is unique among mammalian hexokinases in having a Kmfor glucose in the physiological (8–10 mm) glucose range (6Trus M.D. Zawalich W.S. Burch P.T. Berner D.K. Weill V.A. Matschinsky F.M. Diabetes. 1981; 30: 911-922Crossref PubMed Google Scholar). The high capacity glucose transporter, GLUT2, is also unique in having a high Km (∼17 mm) for glucose (7Thorens B. Deriaz N. Bosco D. DeVos A. Pipeleers D. Schuit F. Meda P. Porret A. J. Biol. Chem. 1996; 271: 8075-8081Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) but is believed to play a more “permissive” role in glucose sensing by allowing the rapid equilibration of glucose across the plasma membrane (8Sweet I.R. Matschinsky F.M. Diabetologia. 1997; 40: 112-119Crossref PubMed Scopus (22) Google Scholar). However, several studies have suggested that the expression of GLUT2 is required for conferring glucose-sensing capabilities into non-β cell lines (9Hughes S.D. Johnson J.H. Quaade C. Newgard C.B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 688-692Crossref PubMed Scopus (115) Google Scholar, 10Motoyoshi S. Shirotani T. Araki E. Sakai K. Kaneko K. Motoshima H. Yoshizato K. Shirakami A. Kishikawa H. Shichiri M. Diabetologia. 1998; 41: 1492-1501Crossref PubMed Scopus (24) Google Scholar). In addition, it has been observed that GLUT2 and GK are coexpressed not only in β cells and hepatocytes but also in glucose-responsive neurons in the hypothalamus and the gut (11Schuit F.C. Huypens P. Heimberg H. Pipeleers D.G. Diabetes. 2001; 50: 1-11Crossref PubMed Scopus (313) Google Scholar), further suggesting that GLUT2 may be an important component of the glucose-sensing apparatus in non-islet cells.It has been suggested that the simple “iterative engineering” of glucose-sensing components into cells or cell lines may simulate the performance of normal islet β cells (12Clark S.A. Quade C. Constandy H. Hansen P. Halban P. Ferber S. Newgard C.B. Normington K. Diabetes. 1997; 46: 958-967Crossref PubMed Scopus (84) Google Scholar). The goal of this study was to determine whether the expression of GK or GLUT2, alone or in combination, would confer glucose-sensing capabilities into ILins cells. We found that the single expression of GK conferred glucose-sensing capabilities in the physiological glucose concentration range and that glycolytic flux was increased by the coexpression of GLUT2. However, unexpectedly, these manipulations also resulted in a state of glucose-induced toxicity with severe apoptotic cell death. These findings have important implications for future strategies to metabolically engineer surrogate β cells.DISCUSSIONWe demonstrate that ILins cells can be engineered to express extremely high levels of functional GK and GLUT2 proteins. The expression of GLUT2 alone enhanced glucose usage only at very low glucose concentrations (i.e. 0.3 mm glucose), similar to a previous report (26Morita H. Yano Y. Niswender K.D. May J.M. Whitesell R.R. Wu L. Printz R.L. Granner D.K. Magnuson M.A. Powers A.C. J. Clin. Invest. 1994; 94: 1373-1382Crossref PubMed Scopus (17) Google Scholar). In contrast, the single expression of GK in ILins cells increased glucose usage and lactate production in the physiological glucose range with a 2-fold increase from 3 to 20 mm glucose, relative to control cells (p < 0.001). These results are consistent with the known regulation of GK enzyme activity that occurs over the same glucose range. The coexpression of GLUT2 and GK further increased glucose usage at 20 mm glucose, but unexpectedly, this was accompanied by a leftward shift in the glucose dose-response curve with disproportionately greater glycolytic flux at 3 mm glucose. Despite the stimulation of glycolysis in these engineered cells, insulin secretion was reduced with a pronounced accumulation of hexose phosphates, depletion of ATP, and apoptotic cell death.Why did glycolysis and energy production go awry in these metabolically engineered cells? One factor may relate to the inherent organization of the glycolytic pathway. The term “turbo design” has been coined to describe the organization of many catabolic pathways that begin with one or more ATP-consuming steps, after which further metabolism and ATP-generating reactions yield a net production of ATP across the pathway (27Teusink B. Walsh M.C. van Dam K. Westerhoff H.V. Trends Biochem. Sci. 1998; 23: 162-169Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). Turbo design is exemplified in glycolysis, in which two molecules of ATP are initially invested at the hexokinase (HK) and phosphofructokinase (PFK) steps as a prelude to the net synthesis of four molecules of ATP further down the pathway. This design feature makes tight regulation of the enzymes involved in the initial ATP-consuming steps absolutely essential (27Teusink B. Walsh M.C. van Dam K. Westerhoff H.V. Trends Biochem. Sci. 1998; 23: 162-169Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). In mammalian cells the activity of HK is inhibited by its product, glucose 6-phosphate (G6P). However, GK is not subject to such feedback inhibition. Therefore, when GK is expressed at high levels, there is the risk of uncontrolled accumulation of G6P, especially at high glucose levels. In addition, because G6P is in equilibrium with fructose 6-phosphate, the fructose 6-phosphate would be expected to rise and increase flux through PFK.In these studies, the levels of F1,6BP were ∼5-fold higher than those of G6P but otherwise showed similar patterns of accumulation. A number of factors may contribute to the preferential accumulation of F1,6BP. First, PFK would be expected to be activated allosterically by the consumption of its inhibitor ATP (and presumably, the corresponding rise in the PFK activator AMP) during glucose phosphorylation. Second, because the product of the PFK reaction, F1,6BP, is itself a potent allosteric activator of PFK, the rise in F1,6BP levels may be autocatalytic. Third, because F1,6BP and glyceraldehyde 3-phosphate (GAP) and dihydroxyacetone phosphate form an equilibrium pool through the rapid aldolase and triose phosphate isomerase reactions, the F1,6BP concentration varies as the square of the GAP concentration (28Tornheim K. J. Theor. Biol. 1979; 79: 491-541Crossref PubMed Scopus (48) Google Scholar), so there may be a disproportionate rise in F1,6BP as glycolytic flux increases. Finally, as more F1,6BP and GAP accumulate, the GAP concentration may approach or surpass the Km of GAPDH, further diminishing the responsiveness of GAPDH to the increased metabolic flux. It should be noted that the maximum accumulated F1,6BP is equivalent to only about 7% of the glucose utilization (or the lactate production), suggesting that there may not be a major block at the reaction catalyzed by GAPDH. However, accumulated F1,6BP is equivalent to nearly twice the normal level of intracellular ATP, and therefore, the sequestration of phosphate in F1,6BP and G6P likely accounts for the drop in cellular ATP levels. Admittedly, our measurements of these metabolites were at the end of 1 h of incubation, and it is possible that these changes did not occur at a constant rate but that the rise in F1,6BP and loss of ATP occurred more acutely toward the end of the incubation period such that phosphate depletion did inhibit the flow through GAPDH (which uses Pias a substrate) and the glycolytic ATP production. Regardless of the exact timing, it is likely that the sequestration of phosphate in G6P and F1,6BP accounts for the ATP depletion and loss of cell viability.It has been suggested that the reason why GK is not hazardous to cell types where it is normally expressed, such as β cells and hepatocytes, is that in the former, GK activity is the lowest of the activities of all glycolytic enzymes and, as such, constitutes the “rate-limiting” step of glycolysis (29Meglasson M.D. Matschinsky F.M. Am. J. Physiol. 1984; 246: E1-E13Crossref PubMed Google Scholar); whereas in hepatocytes, GK is subject to a number of regulatory influences, including the GK regulatory protein. Furthermore, hepatocytes have additional reactions (e.g. glucose cycling through glucose-6-phosphatase and glycogen synthesis) that may prevent the excessive accumulation of glycolytic intermediates (30Iynedjian P.B. Trends Biochem. Sci. 1998; 23: 467-468Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar).A striking feature of the cells that coexpressed GLUT2 and GK was the leftward shift in their glucose-response threshold with a disproportionate increase in glycolytic flux at 3 mmglucose. These findings were unexpected in view of the highKm of GLUT2 and GK and could not be predicted from studying the metabolic impact of each gene individually. A simple explanation is that when GK is expressed alone, endogenous GLUT1 limits the flux of glucose through the cells; but when GLUT2 and GK are coexpressed, neither transport nor phosphorylation is rate-limiting. However, it seems unlikely that glucose transport is rate-limiting, because even in LacZ-infected cells uptake at 3 mm glucose was ∼2-fold higher than the glucose usage in cells coexpressing GLUT2 and GK (10.2 ± 0.9 versus5.6 ± 0.4 nmol/min/mg of protein, respectively). These data suggest that in this setting there may be a direct interaction between GLUT2 and GK (31Hughes S.D. Quaade C. Johnson J.H. Ferber S. Newgard C.B. J. Biol. Chem. 1993; 268: 15205-15212Abstract Full Text PDF PubMed Google Scholar) or, more likely, in combination they may affect the activities of other components involved in the upper part of the glycolytic pathway (32Malaisse W.J. Bodur H. Int. J. Biochem. 1991; 23: 955-959Crossref PubMed Scopus (14) Google Scholar).Similar metabolic perturbations, i.e. activation of the first steps of glycolysis with the accumulation of hexose phosphates and loss of ATP, have been reported in several other systems. These include Saccharomyces cerevisiae yeast mutants that are unable to synthesize the HK inhibitor trehalose 6-phosphate (27Teusink B. Walsh M.C. van Dam K. Westerhoff H.V. Trends Biochem. Sci. 1998; 23: 162-169Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar), trypanosomes in which glycolysis is not compartmentalized (33Bakker B.M. Mensonides F.I. Teusink B. van Hoek P. Michels P.A. Westerhoff H.V. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2087-2092Crossref PubMed Scopus (153) Google Scholar), healthy subjects who received high doses of parenteral fructose, which is phosphorylated by fructokinase that bypasses the regulated HK step (34Gitzelman R. Steinman B. Van den Bergue G. Scriver C.R. Baudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Basis of Inherited Disease. McGraw Hill, Inc., New York1995: 904-934Google Scholar), and the INS-1 β-cell line that was engineered to overexpress large amounts of GK (35Wang H. Iynedjian P.B. J. Biol. Chem. 1997; 272: 25731-25736Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 36Wang H. Iynedjian P.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4372-4377Crossref PubMed Scopus (115) Google Scholar). In the latter setting, control of glycolysis at the GK step was also lost, with a marked increase in glucose usage at low (2.5 mm) glucose concentrations. These effects were attributed to high level GK expression alone (35Wang H. Iynedjian P.B. J. Biol. Chem. 1997; 272: 25731-25736Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), but our findings raise the possibility that the endogenous GLUT2 may have also adversely contributed to this phenotype.Another remarkable feature of GLUT2- and GK-coexpressing cells was that, despite exhibiting an acute 80% drop in intracellular ATP levels at high glucose levels, these cells showed pronounced amounts of apoptotic cell death. These findings were unexpected, because apoptosis is an energyrequiring process and progression to necrotic or apoptotic cell death is thought to depend in part on the cellular ATP content, with rapid ATP depletion usually resulting in necrosis (37Raff M. Nature. 1998; 396: 119-122Crossref PubMed Scopus (589) Google Scholar). These studies suggest that, although the ATP levels in GLUT2/GK-coexpressing cells are severely reduced, these ATP levels are still sufficient to complete the apoptotic program. Recent studies have suggested that glucose may induce apoptosis in pancreatic β cells (38Efanova I. Zaitsev S. Zhivotovsky B. Kohler M. Efendic S. Orrenius S. Berggren P.O. J. Biol. Chem. 1998; 273: 33501-33507Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar, 39Federici M. Hribal M. Perego L. Ranalli M. Caradonna Z. Perego C. Usellini L. Nano R. Bonini P. Bertuzzi F. Marlier L.N.J.L. Davalli A.M. Carandente O. Pontiroli A.E. Melino G. Marchetti P. Lauro R. Sesti G. Folli F. Diabetes. 2001; 50: 1290-1301Crossref PubMed Scopus (270) Google Scholar). Although caution must be used against extrapolating our data to islets, these findings suggest that GLUT2 and GK may be involved in a glucose signaling pathway that, when imbalanced, results in metabolic toxicity and apoptotic cell death.It has been suggested that the simple introduction of glucose-sensing components into cells or cell lines may simulate the performance of normal islet β cells. These findings raise important caveats to this notion and demonstrate the deleterious consequences that can result when the expression levels of the metabolic components are not closely optimized. We must emphasize that the metabolic toxicity described in this report was not due to the high level expression achieved with this particular viral gene delivery system. We have recently created a series of transgenic mouse lines that coexpress GLUT2 and GK in IL cells at levels that are markedly lower than those achieved with adenoviruses. Interestingly, the size of the IL tissues in these transgenic mice corresponded inversely to the levels of GK expression, with the most severe reduction in the size of the IL tissues in the lines that expressed the highest levels of GK. 2R. N. Faradji, E. Havari, Q. Chen, and M. A. Lipes, manuscript in preparation. Although the mechanism by which glucose stimulates insulin secretion in β cells is complex and clearly requires more than just the expression of GLUT2 and GK, these transgenic tissues should provide an excellent starting material for determining the requirements for optimal glucose-sensing in surrogate β cells. Type 1 diabetes in humans and in nonobese diabetic (NOD)1 mice is an autoimmune disorder that results in the complete destruction of the insulin-producing pancreatic β cells. Despite recent advances in insulin injection therapy, current regimens do not adequately mimic the normal physiological patterns of insulin release by β cells, and patients with diabetes are thus at continued risk for developing severe long-term complications. These limitations have provided the impetus to develop alternative forms of therapy that would circumvent the need for insulin injections. One appealing therapeutic strategy is to engineer insulin expression into non-β cells (1Lipes M.A. Cooper E.M. Skelly R. Rhodes C.J. Boschetti E. Weir G.C. Davalli A.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8595-8600Crossref PubMed Scopus (78) Google Scholar, 2Chen R. Meseck M. McEvoy R.C. Woo S.L. Gene Ther. 2000; 7: 1802-1809Crossref PubMed Scopus (56) Google Scholar, 3Cheung A.T. Dayanandan B. Lewis J.T. Korbutt G.S. Rajotte R.V. Bryer-Ash M. Boylan M.O. Wolfe M.M. Keiffer T.J. Science. 2000; 290: 1959-1962Crossref PubMed Scopus (256) Google Scholar). Using transgenic mouse techniques, we targeted mouse preproinsulin 2 (ins) gene expression to the intermediate lobe (IL) of the pituitary in NOD mice via the proopiomelanocortin (POMC) promoter (1Lipes M.A. Cooper E.M. Skelly R. Rhodes C.J. Boschetti E. Weir G.C. Davalli A.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8595-8600Crossref PubMed Scopus (78) Google Scholar). Our studies showed that the IL cells secreted large amounts of insulin, sufficient to cure diabetes when implanted into spontaneously diabetic NOD mice. Moreover, in contrast to transplanted islets, the insulin-producing IL (hereafter, ILins) cells evaded immune recognition and damage. These studies were the first to show that a non-islet cell type could be engineered to secrete sufficient amounts of insulin to cure diabetes, yet escape the autoimmune process that kills β cells in type 1 diabetes (1Lipes M.A. Cooper E.M. Skelly R. Rhodes C.J. Boschetti E. Weir G.C. Davalli A.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8595-8600Crossref PubMed Scopus (78) Google Scholar). However, a limitation of this insulin gene delivery system was that insulin secretion was not glucose-regulated. The regulation of insulin release in β cells is mediated by the metabolism of glucose (4Meglasson M.D. Matschinsky F.M. Diabetes Metab. Rev. 1986; 2: 163-214Crossref PubMed Scopus (412) Google Scholar). According to current models, glucose metabolism generates signals such as ATP or an increase in the ratio of ATP to ADP ratio that leads to closure of the ATP-sensitive K+ (KATP) channels. The resulting plasma membrane depolarization activates voltage-gated L-type Ca2+channels, inducing the influx of Ca2 that triggers insulin granule exocytosis (5Newgard C.B. McGarry J.D. Annu. Rev. Biochem. 1995; 64: 689-719Crossref PubMed Scopus (491) Google Scholar). The proteins that control the metabolic flux of glucose in β cells function as “glucose sensors.” Rodent β cells express two specialized proteins that have been considered as candidates for the glucose sensor: the facilitated glucose transporter isoform, GLUT2, and the low affinity glucose phosphorylating enzyme, glucokinase (GK). A large body of evidence suggests that GK is the flux-controlling enzyme for glycolysis in β cells and, as such, serves as the “gatekeeper” for metabolic signaling (4Meglasson M.D. Matschinsky F.M. Diabetes Metab. Rev. 1986; 2: 163-214Crossref PubMed Scopus (412) Google Scholar). GK is unique among mammalian hexokinases in having a Kmfor glucose in the physiological (8–10 mm) glucose range (6Trus M.D. Zawalich W.S. Burch P.T. Berner D.K. Weill V.A. Matschinsky F.M. Diabetes. 1981; 30: 911-922Crossref PubMed Google Scholar). The high capacity glucose transporter, GLUT2, is also unique in having a high Km (∼17 mm) for glucose (7Thorens B. Deriaz N. Bosco D. DeVos A. Pipeleers D. Schuit F. Meda P. Porret A. J. Biol. Chem. 1996; 271: 8075-8081Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) but is believed to play a more “permissive” role in glucose sensing by allowing the rapid equilibration of glucose across the plasma membrane (8Sweet I.R. Matschinsky F.M. Diabetologia. 1997; 40: 112-119Crossref PubMed Scopus (22) Google Scholar). However, several studies have suggested that the expression of GLUT2 is required for conferring glucose-sensing capabilities into non-β cell lines (9Hughes S.D. Johnson J.H. Quaade C. Newgard C.B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 688-692Crossref PubMed Scopus (115) Google Scholar, 10Motoyoshi S. Shirotani T. Araki E. Sakai K. Kaneko K. Motoshima H. Yoshizato K. Shirakami A. Kishikawa H. Shichiri M. Diabetologia. 1998; 41: 1492-1501Crossref PubMed Scopus (24) Google Scholar). In addition, it has been observed that GLUT2 and GK are coexpressed not only in β cells and hepatocytes but also in glucose-responsive neurons in the hypothalamus and the gut (11Schuit F.C. Huypens P. Heimberg H. Pipeleers D.G. Diabetes. 2001; 50: 1-11Crossref PubMed Scopus (313) Google Scholar), further suggesting that GLUT2 may be an important component of the glucose-sensing apparatus in non-islet cells. It has been suggested that the simple “iterative engineering” of glucose-sensing components into cells or cell lines may simulate the performance of normal islet β cells (12Clark S.A. Quade C. Constandy H. Hansen P. Halban P. Ferber S. Newgard C.B. Normington K. Diabetes. 1997; 46: 958-967Crossref PubMed Scopus (84) Google Scholar). The goal of this study was to determine whether the expression of GK or GLUT2, alone or in combination, would confer glucose-sensing capabilities into ILins cells. We found that the single expression of GK conferred glucose-sensing capabilities in the physiological glucose concentration range and that glycolytic flux was increased by the coexpression of GLUT2. However, unexpectedly, these manipulations also resulted in a state of glucose-induced toxicity with severe apoptotic cell death. These findings have important implications for future strategies to metabolically engineer surrogate β cells. DISCUSSIONWe demonstrate that ILins cells can be engineered to express extremely high levels of functional GK and GLUT2 proteins. The expression of GLUT2 alone enhanced glucose usage only at very low glucose concentrations (i.e. 0.3 mm glucose), similar to a previous report (26Morita H. Yano Y. Niswender K.D. May J.M. Whitesell R.R. Wu L. Printz R.L. Granner D.K. Magnuson M.A. Powers A.C. J. Clin. Invest. 1994; 94: 1373-1382Crossref PubMed Scopus (17) Google Scholar). In contrast, the single expression of GK in ILins cells increased glucose usage and lactate production in the physiological glucose range with a 2-fold increase from 3 to 20 mm glucose, relative to control cells (p < 0.001). These results are consistent with the known regulation of GK enzyme activity that occurs over the same glucose range. The coexpression of GLUT2 and GK further increased glucose usage at 20 mm glucose, but unexpectedly, this was accompanied by a leftward shift in the glucose dose-response curve with disproportionately greater glycolytic flux at 3 mm glucose. Despite the stimulation of glycolysis in these engineered cells, insulin secretion was reduced with a pronounced accumulation of hexose phosphates, depletion of ATP, and apoptotic cell death.Why did glycolysis and energy production go awry in these metabolically engineered cells? One factor may relate to the inherent organization of the glycolytic pathway. The term “turbo design” has been coined to describe the organization of many catabolic pathways that begin with one or more ATP-consuming steps, after which further metabolism and ATP-generating reactions yield a net production of ATP across the pathway (27Teusink B. Walsh M.C. van Dam K. Westerhoff H.V. Trends Biochem. Sci. 1998; 23: 162-169Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). Turbo design is exemplified in glycolysis, in which two molecules of ATP are initially invested at the hexokinase (HK) and phosphofructokinase (PFK) steps as a prelude to the net synthesis of four molecules of ATP further down the pathway. This design feature makes tight regulation of the enzymes involved in the initial ATP-consuming steps absolutely essential (27Teusink B. Walsh M.C. van Dam K. Westerhoff H.V. Trends Biochem. Sci. 1998; 23: 162-169Abstract Full Text Full Text PDF Pub" @default.
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• W1965867110 title "Glucose-induced Toxicity in Insulin-producing Pituitary Cells That Coexpress GLUT2 and Glucokinase" @default.
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