Pancreatic β-Cell-specific Targeted Disruption of Glucokinase Gene
1995; Elsevier BV; Volume: 270; Issue: 51 Linguagem: Inglês
10.1074/jbc.270.51.30253
ISSN1083-351X
AutoresYasuo Terauchi, Hiroshi Sakura, Kazuki Yasuda, Keiji Iwamoto, Noriko Takahashi, Kouichi Ito, Haruo Kasai, Hiroshi Suzuki, Otoya Ueda, Nobuo Kamada, Kou‐ichi Jishage, Kajuro Komeda, Mitsuhiko Noda, Yasunori Kanazawa, Shigeki Taniguchi, Ichitomo Miwa, Yasuo Akanuma, Tatsuhiko Kodama, Yoshio Yazaki, Takashi Kadowaki,
Tópico(s)Diabetes and associated disorders
ResumoMice carrying a null mutation in the glucokinase (GK) gene in pancreatic β-cells, but not in the liver, were generated by disrupting the β-cell-specific exon. Heterozygous mutant mice showed early-onset mild diabetes due to impaired insulin-secretory response to glucose. Homozygotes showed severe diabetes shortly after birth and died within a week. GK-deficient islets isolated from homozygotes showed defective insulin secretion in response to glucose, while they responded to other secretagogues: almost normally to arginine and to some extent to sulfonylureas. These data provide the first direct proof that GK serves as a glucose sensor molecule for insulin secretion and plays a pivotal role in glucose homeostasis. GK-deficient mice serve as an animal model of the insulin-secretory defect in human non-insulin-dependent diabetes mellitus. Mice carrying a null mutation in the glucokinase (GK) gene in pancreatic β-cells, but not in the liver, were generated by disrupting the β-cell-specific exon. Heterozygous mutant mice showed early-onset mild diabetes due to impaired insulin-secretory response to glucose. Homozygotes showed severe diabetes shortly after birth and died within a week. GK-deficient islets isolated from homozygotes showed defective insulin secretion in response to glucose, while they responded to other secretagogues: almost normally to arginine and to some extent to sulfonylureas. These data provide the first direct proof that GK serves as a glucose sensor molecule for insulin secretion and plays a pivotal role in glucose homeostasis. GK-deficient mice serve as an animal model of the insulin-secretory defect in human non-insulin-dependent diabetes mellitus. INTRODUCTIONGlucokinase (GK), 1The abbreviations used are: GKglucokinaseNIDDMnon-insulin-dependent diabetes mellitusES cellembryonic stem cellHKhexokinaseHanks'-BSA bufferHanks'-buffered saline containing 0.2% bovine serum albumin. mainly expressed in pancreatic β-cells and the liver, is thought to constitute a rate-limiting step in glucose metabolism in these tissues (1.Matschinsky F.M. Diabetes. 1990; 39: 647-652Crossref PubMed Google Scholar, 2.Magnuson M. Diabetes. 1990; 39: 523-527Crossref PubMed Google Scholar, 3.Matschinsky F. Liang Y. Kesavan P. Wang L. Froguel P. Velho G. Cohen D. Permutt M.A. Tanizawa Y. Jetton T.L. Niswender K. Magnuson M.A. J. Clin. Invest. 1993; 92: 2092-2098Crossref PubMed Scopus (257) Google Scholar, 4.Pilkis S.J. Weber I.T. Harrison R.W. Bell G.I. J. Biol. Chem. 1994; 269: 21925-21928Abstract Full Text PDF PubMed Google Scholar). Since insulin secretion parallels glucose metabolism and the high Km of GK (5-8 mM) ensures that it can change its enzymatic activity within the physiological range of glucose concentrations, GK has been proposed to act as a glucose sensor in the pancreatic β-cell(1.Matschinsky F.M. Diabetes. 1990; 39: 647-652Crossref PubMed Google Scholar, 5.Randle P.J. Diabetologia. 1993; 36: 269-275Crossref PubMed Scopus (71) Google Scholar). Recently, mutations of the GK gene have been identified in patients with maturity-onset diabetes of the young, a subtype of early-onset non-insulin-dependent diabetes mellitus (NIDDM)(6.Vionnet N. Stoffel M. Takeda J. Yasuda K. Bell G.I. Zouali H. Lesage S. Velho G. Iris F. Passa Ph. Floguel Ph. Cohen D. Nature. 1992; 356: 721-722Crossref PubMed Scopus (554) Google Scholar, 7.Katagiri H. Asano T. Ishihara H. Inukai K. Anai M. Miyazaki J. Tsukuda K. Kikuchi M. Yazaki Y. Oka Y. Lancet. 1992; 340: 1316-1317Abstract Full Text PDF PubMed Scopus (75) Google Scholar, 8.Sakura H. Etoh K. Kadowaki H. Shimokawa K. Ueno H. Koda N. Fukushima Y. Akanuma Y. Yazaki Y. Kadowaki T. J. Clin. Endocrinol. Metab. 1992; 75: 1571-1573PubMed Google Scholar). However, since all the mutations in humans so far occur in the region of the gene that is common to pancreatic β-cells and hepatocytes(9.Magnuson M.A. Shelton K.D. J. Biol. Chem. 1989; 264: 15936-15942Abstract Full Text PDF PubMed Google Scholar), and are heterozygous, it may not have been possible to fully reveal physiological roles of pancreatic β-cell GK either in vivo or in vitro. To this end, mice carrying a null mutation in the GK gene in pancreatic β-cells, but not in the liver, were generated by homologous recombination. Heterozygous mutant mice showed early-onset mild diabetes resembling the phenotype for human maturity-onset diabetes of the young. Homozygotes showed severe diabetes shortly after birth and died within a week. GK-deficient islets showed defective insulin secretion in response to glucose, while they responded to other secretagogues: almost normally to arginine and to some extent to sulfonylureas. These data provide the first direct proof that GK serves as a glucose sensor molecule for insulin secretion and plays a pivotal role in glucose homeostasis.EXPERIMENTAL PROCEDURESCloning of the Mouse GK Gene, Construction of a Targeting Vector, and Homologous Recombinant ExperimentsA DNA fragment including the pancreatic β-cell-specific exon 1β of the GK gene was cloned from a BALB/c mouse genomic library (Clontech). A BamHI site was introduced 30 base pairs 3′ to the translation initiation codon of GK by the Kunkel method(10.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). A neomycin resistance gene (neor) with a pgk-1 promoter but without a poly(A)+ addition signal was substituted for the XbaI-BamHI fragment in the exon 1β. A diphtheria toxin A fragment gene (DTA) with a MC1 promoter was ligated on the 3′ terminus across the vector backbone, for negative selection(11.Yagi T. Aizawa S. Tokunaga T. Shigetani Y. Takeda N. Ikawa Y. Nature. 1993; 366: 742-745Crossref PubMed Scopus (140) Google Scholar, 12.Tamemoto H. Kadowaki T. Tobe K. Yagi T. Sakura H. Hayakawa T. Terauchi Y. Ueki K. Kaburagi Y. Satoh S. Sekihara H. Yoshioka S. Horikoshi H. Furuta Y. Ikawa Y. Kasuga M. Yazaki Y. Aizawa S. Nature. 1994; 372: 182-186Crossref PubMed Scopus (898) Google Scholar). Homologous recombinant experiments in embryonic stem cells (ES cells) (A3-1) (13.Azuma S. Toyoda Y. Jpn. J. Anim. Reprod. 1991; 37: 37-43Crossref Google Scholar, 14.Kurihara Y. Kurihara H. Suzuki H. Kodama T. Maemura K. Nagai R. Oda H. Kuwaki T. Cao W.H. Kamada N. Jishage K. Ouchi Y. Azuma S. Toyoda Y. Ishikawa T. Kumada N. Yazaki Y. Nature. 1994; 368: 703-710Crossref PubMed Scopus (886) Google Scholar) were carried out as described previously(14.Kurihara Y. Kurihara H. Suzuki H. Kodama T. Maemura K. Nagai R. Oda H. Kuwaki T. Cao W.H. Kamada N. Jishage K. Ouchi Y. Azuma S. Toyoda Y. Ishikawa T. Kumada N. Yazaki Y. Nature. 1994; 368: 703-710Crossref PubMed Scopus (886) Google Scholar). These cells were injected into blastocysts from C57BL/6J mice or co-cultured with morulae from C57BL/6J mice(15.Wood S.A. Pascoe W.S. Schmidt C. Kemler R. Evans M.J. Allen N.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4582-4585Crossref PubMed Scopus (96) Google Scholar, 16.Suzuki H. Kamada N. Ueda O. Jishage K. Kurihara H. Kurihara Y. Kodama T. Yazaki Y. Azuma S. Toyoda Y. J. Reprod. Dev. 1994; 40: 361-365Crossref Scopus (9) Google Scholar) and transferred into pseudopregnant ICR females to generate offspring.Determination of Glucose Phosphorylating ActivityIslets were isolated from the pancreas of 3-7-day-old mice by collagenase digestion method(17.Gotoh M. Maki T. Kiyoizumi T. Satomi S. Monaco A.P. Transplantation. 1985; 40: 437-438Crossref PubMed Scopus (535) Google Scholar). Glucose phosphorylating activities by hexokinase (HK) and GK were determined fluorometrically (18.Miwa, I., Mita, Y., Murata, T., Okuda, J., Sugiura, M., Hamada, Y., Chiba, T. (1995) Enzyme & Protein, in pressGoogle Scholar) except that GK activity was measured at 50 mM glucose.Glucose Tolerance TestMice (10 weeks old) were fasted for more than 16 h before the study. They were then loaded with 1.5 mg g−1−1 (body weight) glucose by intraperitoneal infusion. Blood samples were taken at different time points from the orbital sinus. Insulin levels were determined using an insulin radioimmunoassay kit (Shionogi) with rat insulin as standard.ImmunohistochemistryPancreata were immersion-fixed in 4.0% (w/v) paraformaldehyde, 0.1 M sodium phosphate buffer at 4°C overnight. Diluted guinea pig anti-porcine insulin (DAKO, A564) (1:200), or rabbit anti-porcine glucagon (DAKO, A565) (1:200), or rabbit anti-human somatostatin (DAKO, A566) (1:200) was applied to the sections for 45 min at room temperature. The sections were then rinsed with Tris-buffered saline, and then treated with a second antibody.Insulin Content AssayIsolated islets were suspended in 100 μl of acid ethanol, and cellular insulin was extracted, diluted (100 times), and assayed by radioimmunoassay.Determination of Intracellular Calcium ConcentrationIsolated islets were incubated overnight with RPMI 1640 medium and measurements of intracellular calcium concentration were carried out using fura-2 acetoxymethylester (Molecular Probes) (19.Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar, 20.Weinhaus A.J. Poronnik P. Cook D.I. Tuch B.E. Diabetes. 1995; 44: 118-124Crossref PubMed Scopus (43) Google Scholar) by the method of Weinhaus et al.(20.Weinhaus A.J. Poronnik P. Cook D.I. Tuch B.E. Diabetes. 1995; 44: 118-124Crossref PubMed Scopus (43) Google Scholar).Batch Incubation StudyBatches of 10 islets were preincubated for 60 min at 37°C in 5% CO2 in 1 ml of Hank's-buffered saline containing 0.2% bovine serum albumin, 10 mM HEPES, pH 7.5 (Hanks'-BSA buffer) plus 0.1 mM glucose. The medium was then replaced with 1 ml of Hanks'-BSA buffer supplemented with secretagogues. After a 60-min incubation at 37°C, the medium was removed for radioimmunoassay of insulin (Shionogi).RESULTS AND DISCUSSIONTargeted Disruption of Glucokinase Gene in Pancreatic β-CellsAlternative splicing of a single GK gene gives rise to two isoforms of GK, one expressed in the pancreatic β-cells and the other in liver, which have different first exons (exon 1β and exon 1L, respectively) (Fig. 1A)(9.Magnuson M.A. Shelton K.D. J. Biol. Chem. 1989; 264: 15936-15942Abstract Full Text PDF PubMed Google Scholar). These two isoforms are transcribed by two different promoters, and the downstream promoter, which lies between exon 1β and 1L, drives transcription of the liver GK isoform. We were therefore able to disrupt exon 1β expression and thereby selectively eliminate expression of the pancreatic β-cell isoform of GK without affecting expression of the liver isoform. Eight independent ES cell clones were identified as carrying the targeted mutant GK allele (Fig. 1B). Male chimeras originated from two homologous recombinant clones transmitted the mutant GK allele to their offspring. Heterozygous mutant mice were apparently normal and gave birth to mice homozygous for the mutant GK allele. The ratio of wild-type, heterozygous, and homozygous mice was 80:170:85 in 335 offsprings at 3-4 days of age, which was consistent with Mendelian inheritance (Fig. 1B). Reverse-transcriptase PCR analysis revealed that GK expression in pancreatic β-islets was completely absent in homozygotes (data not shown). In islets from homozygous neonates, GK activity was completely absent, whereas HK activity was similar to that from wild-type neonates (Fig. 1C). In islets from adult heterozygotes, GK activity (Vmax) was 48% of that from the wild-type mice. In contrast to the β-cell, both GK and HK activities in the liver were unaltered in each genotype (data not shown).Mild Diabetes in Heterozygous GK Knock-out MiceAt birth, the blood glucose levels of heterozygous and wild-type mice were 2.5 ± 0.3 (n = 5), and 2.4 ± 0.1 (n = 4) mM, respectively. However, about 50% of the heterozygous mice showed mild glycosuria within a day, suggesting the development of early-onset diabetes mellitus. Heterozygous mice (10 weeks old) showed significantly higher blood glucose levels both before and after a glucose load and smaller increments in serum insulin levels than wild-type mice (Fig. 2). Insulin tolerance test revealed that the heterozygous mutant mice were as sensitive to insulin as the wild-type (data not shown). These results demonstrate that a heterozygous mutation of the GK gene in the pancreatic β-cells is sufficient to cause impaired insulin secretion to glucose and diabetes mellitus. In this respect, Efrat et al.(21.Efrat S. Leiser M. Wu Y. Fusco-DeMane D. Emran O. Surana M. Jetton T.L. Magnuson M.A. Weir G. Fleischer N. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2051-2055Crossref PubMed Scopus (113) Google Scholar) have generated mice in which pancreatic GK expression was attenuated by a ribozyme-mediated method. Although GK activity of these mice was only 30% that of normal and they showed an impaired insulin response to glucose in perfused pancreas experiments, the fasting and postprandial glucose levels remained normal. It is possible that the different strategies used to attenuate GK expression or the different strains of mice used (Efrat et al. used C3H, whereas this report used ICR) may explain the apparent discrepancy between their and our results.Fig. 2Mild diabetes in heterozygous GK knock-out mice. Results of a glucose tolerance test are shown. Wild-type (open circles) and heterozygous mice (open triangles) were loaded with 1.5 mg g (body weight) glucose, and blood glucose (upper panel) and serum insulin (lower panel) levels were determined at the indicated time points. The data points indicate mean ± S.E. (n = 15).∗∗, p < 0.01; ∗, p < 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Characterization of Homozygous GK Knock-out MiceHomozygous mutant pups were normal in size, appearance, and body weight at birth (Fig. 3A). Their blood glucose level was 2.7 ± 0.1 mM (n = 4), which was indistinguishable from that of wild-type mice (2.4 ± 0.1 mM, n = 4). Although they were able to suck as evidenced by the presence of milk in their stomachs, they showed no increase in body weight with age (Fig. 3A). They also showed marked glycosuria within a day, and almost all animals died within 7 days of birth apparently due to dehydration. At 3-4 days of age, their blood glucose levels were markedly higher than those of wild-type or heterozygous animals, while serum insulin levels were low relative to the elevated blood glucose concentrations, suggesting the presence of relative insulin deficiency (Fig. 3B). Only 20% of homozygotes showed ketosis in spite of marked hyperglycemia. These suggest that basal insulin secretion is preserved in homozygotes presumably due to activity of the β-cell HK. Thus, diabetes mellitus in the GK-deficient mice, although severe, is more similar to NIDDM rather than insulin-dependent diabetes mellitus. Post-mortem investigation revealed no gross abnormalities in any of the organs examined except for occasional fatty changes of the liver in homozygous mutants (data not shown).Fig. 3Characterization of homozygous GK knock-out mice. A, changes in body weight of neonates. The genotype of the neonates was determined by Southern blot analysis (Fig. 1B). The body weight of the wild-type (open circles), the heterozygotes (open triangles), the homozygotes (crosses) is plotted against the number of days after birth.∗∗, p < 0.01; ∗, p < 0.05 compared with the wild-type. B, relationship between the blood glucose levels and the serum insulin levels at 3-4 days of age. Mice of each genotype were fed freely, and blood samples were collected by decapitation.View Large Image Figure ViewerDownload Hi-res image Download (PPT)9 out of 10 homozygous mutant pups subcutaneously injected with 10-20 milliunits of human insulin (Novolin R, Novo) twice a day survived beyond 7 days of age, and 4 out of 6 homozygous mutant pups orally administered with 20 μmol of glibenclamide (kindly provided by Yamanouchi Pharmaceutical Co.) per day survived beyond 10 days of age, while all untreated pups (n = 50) died before 7 days of age. Both of these treatments significantly lowered blood glucose levels (by 20-40%) and caused a gain in body weight (to approximately 80% of the wild-type littermate). Since GK is also expressed in rare neuroendocrine cells in the brain and gut(22.Jetton T.L. Liang Y. Pethepher C.C. Zimmerman E.C. Cox F.G. Horvath K. Matschinsky F.M. Magnuson M.A. J. Biol. Chem. 1994; 269: 3641-3654Abstract Full Text PDF PubMed Google Scholar), it is possible that the absence of glucose sensing in the brain or gut by GK may have modulated the phenotype of homozygous mutant pups. Nevertheless, it seems likely that hyperglycemia due to a lack of β-cell GK is the major cause of severe metabolic failure and early death in several days, since insulin or sulfonylurea improved these features. Immunostaining for glucagon, insulin, and somatostatin revealed that differentiation into pancreatic α, β, and δ cells also appeared to be unaffected (Fig. 4). Although there may be subtle changes in the architecture of α, β, and δ cells in the islets, its gross appearance was normal. The insulin contents of the wild-type, heterozygous, and homozygous GK-deficient islets were 1.7 ± 0.3 (n = 5), 2.2 ± 0.7 (n = 6), and 2.5 ± 0.2 (n = 10) ng of insulin/islet, respectively. These data indicate that β-cell GK is not essential for normal development, differentiation of endocrine pancreas, or insulin biosynthesis.Fig. 4Immunohistochemistry of GK-deficient islets. Islets were stained for insulin (a and d), glucagon (b and e), or somatostatin (c and f). a-c are the same section of an islet from a wild-type mouse, while d-f are the same section of an islet from a homozygous mutant mouse. Scale bar, 100 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Characterization of GK-deficient IsletsImpact of lack of GK in the pancreatic β-cell was investigated in isolated islets from 7-10 days old mice. A rise in the intracellular Ca2+ concentration in the β-cell ([Ca2+]i) is a key event in glucose-stimulated insulin secretion(19.Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar, 20.Weinhaus A.J. Poronnik P. Cook D.I. Tuch B.E. Diabetes. 1995; 44: 118-124Crossref PubMed Scopus (43) Google Scholar). Although all the GK-deficient islets looked alike under microscopy, they could be subdivided into two groups in terms of basal [Ca2+]i levels; those with normal calcium levels (110 ± 34 nM, n = 7; about 20% of the islets), and those with higher basal calcium levels (higher than 200 nM; about 80%) which might reflect some damage of the islets. In the former group, the increase in [Ca2+]i elicited by glucose was completely abolished, while the increase evoked by glibenclamide or arginine was essentially normal (Fig. 5A). However, in islets with higher basal calcium levels, the increase in [Ca2+]i elicited not only by glucose but also by glibenclamide was completely abolished and that by arginine modestly impaired (data not shown). The observed heterogeneity of GK-deficient islets may be due to a direct effect of the impaired glucose metabolism in GK-deficient islets or may be a consequence of the hyperglycemia and other metabolic defects. Even in wild-type or heterozygous GK-deficient islets, there were islets with higher basal calcium levels (more than 200 nM), but its proportion was less than 10%.Fig. 5Characterization of GK-deficient islets. A, increase in intracellular calcium concentration of islets elicited by 20 mM glucose, 10 μM glibenclamide, or 20 mM arginine. Basal calcium levels in the wild-type, heterozygous, and homozygous islets are 78 ± 15, 116 ± 30, and 110 ± 34 nM, respectively. Values are expressed in nM, as mean ± S.E. (n = 4-7). B, insulin secretion in response to the indicated secretagogues. Values are expressed in nanograms of insulin 10 islets−1 h, as mean ± S.E. (n = 6-15). Solid bar denotes the wild-type (Wild), hatched bar the heterozygotes (Hetero), and open bar the homozygotes (Null). Insulin secretion in response to 3 or 10 mM glucose from null islets was not determined. ∗∗, p < 0.01; ∗, p < 0.05 compared with the wild-type.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We next examined insulin secretion using the batch incubation method (Fig. 5B). Insulin secretion from heterozygous GK-deficient islets in response to 0.1 mM or 3 mM glucose was normal, but that in response to 10 mM glucose was significantly impaired compared with wild-type islets. Impairment in insulin secretion in response to 20 mM glucose was less evident. On the other hand, insulin secretion in response to glibenclamide or arginine was unaffected. In homozygous GK-deficient islets, although there was some basal insulin secretion at 0.1 mM glucose, presumably due to the activity of HK, increase in insulin secretion in response to 20 mM glucose was completely abolished. In contrast, insulin secretion in response to arginine was essentially preserved. Regarding insulin secretion in response to glibenclamide, it was decreased by about 50-80% depending on the method of estimation (Fig. 5B). Nevertheless, since the islets used here were supposed to be a mixture of those with normal and those with higher basal calcium levels (expected to be 20% and 80% of the population, respectively), which were responsive and unresponsive to glibenclamide in calcium study, we interpreted these results as suggesting that islets with normal basal calcium levels may have responded to sulfonylurea in insulin secretion.The insulin secretory response to a physiological increment in glucose concentration was impaired in heterozygous GK-deficient islets and completely defective in homozygous GK-deficient islets despite the presence of HK, supporting the concept that GK serves as a glucose sensor molecule for insulin secretion. This is consistent with the smaller increments in serum insulin levels after a glucose load in heterozygous mice (Fig. 2), lack of increments in insulin levels in homozygous mice in spite of hyperglycemia (Fig. 3B), and the secretory abnormalities in human subjects with GK mutations(23.Byrne M.M. Sturis J. Clement K. Vionnet N. Pueyo M.E. Stoffel M. Takeda J. Passa P. Cohen D. Bell G.I. Velho G. Froguel P. Polonsky K.S. J. Clin. Invest. 1994; 93: 1120-1130Crossref PubMed Scopus (263) Google Scholar). GK-deficient islets responded to non-glucose secretagogues in insulin secretion (almost normally to arginine and to some extent to sulfonylureas), indicating that GK is not absolutely required for insulin secretion in response to these secretagogues. It should also be noted that insulin secretion in response to glibenclamide was impaired, suggesting that GK may play an important role in insulin secretion in response to some of non-glucose secretagogues such as glibenclamide. This possibility would be examined in future. The heterozygous or insulin-treated homozygous mutant mice described here provide the first animal model of diabetes with a defined genetic defect in insulin secretion, and should give important insights into the pathogenesis and development of human NIDDM. INTRODUCTIONGlucokinase (GK), 1The abbreviations used are: GKglucokinaseNIDDMnon-insulin-dependent diabetes mellitusES cellembryonic stem cellHKhexokinaseHanks'-BSA bufferHanks'-buffered saline containing 0.2% bovine serum albumin. mainly expressed in pancreatic β-cells and the liver, is thought to constitute a rate-limiting step in glucose metabolism in these tissues (1.Matschinsky F.M. Diabetes. 1990; 39: 647-652Crossref PubMed Google Scholar, 2.Magnuson M. Diabetes. 1990; 39: 523-527Crossref PubMed Google Scholar, 3.Matschinsky F. Liang Y. Kesavan P. Wang L. Froguel P. Velho G. Cohen D. Permutt M.A. Tanizawa Y. Jetton T.L. Niswender K. Magnuson M.A. J. Clin. Invest. 1993; 92: 2092-2098Crossref PubMed Scopus (257) Google Scholar, 4.Pilkis S.J. Weber I.T. Harrison R.W. Bell G.I. J. Biol. Chem. 1994; 269: 21925-21928Abstract Full Text PDF PubMed Google Scholar). Since insulin secretion parallels glucose metabolism and the high Km of GK (5-8 mM) ensures that it can change its enzymatic activity within the physiological range of glucose concentrations, GK has been proposed to act as a glucose sensor in the pancreatic β-cell(1.Matschinsky F.M. Diabetes. 1990; 39: 647-652Crossref PubMed Google Scholar, 5.Randle P.J. Diabetologia. 1993; 36: 269-275Crossref PubMed Scopus (71) Google Scholar). Recently, mutations of the GK gene have been identified in patients with maturity-onset diabetes of the young, a subtype of early-onset non-insulin-dependent diabetes mellitus (NIDDM)(6.Vionnet N. Stoffel M. Takeda J. Yasuda K. Bell G.I. Zouali H. Lesage S. Velho G. Iris F. Passa Ph. Floguel Ph. Cohen D. Nature. 1992; 356: 721-722Crossref PubMed Scopus (554) Google Scholar, 7.Katagiri H. Asano T. Ishihara H. Inukai K. Anai M. Miyazaki J. Tsukuda K. Kikuchi M. Yazaki Y. Oka Y. Lancet. 1992; 340: 1316-1317Abstract Full Text PDF PubMed Scopus (75) Google Scholar, 8.Sakura H. Etoh K. Kadowaki H. Shimokawa K. Ueno H. Koda N. Fukushima Y. Akanuma Y. Yazaki Y. Kadowaki T. J. Clin. Endocrinol. Metab. 1992; 75: 1571-1573PubMed Google Scholar). However, since all the mutations in humans so far occur in the region of the gene that is common to pancreatic β-cells and hepatocytes(9.Magnuson M.A. Shelton K.D. J. Biol. Chem. 1989; 264: 15936-15942Abstract Full Text PDF PubMed Google Scholar), and are heterozygous, it may not have been possible to fully reveal physiological roles of pancreatic β-cell GK either in vivo or in vitro. To this end, mice carrying a null mutation in the GK gene in pancreatic β-cells, but not in the liver, were generated by homologous recombination. Heterozygous mutant mice showed early-onset mild diabetes resembling the phenotype for human maturity-onset diabetes of the young. Homozygotes showed severe diabetes shortly after birth and died within a week. GK-deficient islets showed defective insulin secretion in response to glucose, while they responded to other secretagogues: almost normally to arginine and to some extent to sulfonylureas. These data provide the first direct proof that GK serves as a glucose sensor molecule for insulin secretion and plays a pivotal role in glucose homeostasis.
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