Pancreatic β-Cell-specific Targeted Disruption of Glucokinase Gene

Abstract

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. 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 is more similar to than diabetes mellitus. revealed no in of the except for of the liver in homozygous (data not of homozygous GK knock-out mice. in body weight of The genotype of the neonates was determined by analysis (Fig. 1B). The body weight of the wild-type (open the (open the homozygotes is the of days after p < 0.01; ∗, p < with the between the blood glucose levels and the serum insulin levels at 3-4 days of Mice of each genotype were and blood samples were by Large Image Figure ViewerDownload Hi-res image Download out of 10 homozygous mutant pups injected with of human insulin a 7 days of age, and out of homozygous mutant pups with of by 10 days of age, while all pups (n = died before 7 days of of these significantly blood glucose levels and a in body weight of the wild-type Since GK is also expressed in cells in the and T.L. Liang Y. K. F.M. Magnuson M.A. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar), it is possible that the of glucose in the or by GK may have the phenotype of homozygous mutant it that due to a of β-cell GK is the cause of severe and in since insulin or these for and somatostatin revealed that into pancreatic and cells also to (Fig. 4). Although may in the of and cells in the its was normal. The insulin of the wild-type, heterozygous, and homozygous GK-deficient islets were ± 0.3 (n = 5), ± (n = and 2.5 ± (n = of respectively. These data indicate that β-cell GK is not for normal of or insulin of GK-deficient were for insulin and glucagon and or somatostatin and are the of an from a wild-type while are the of an from a homozygous mutant 100 Large Image Figure ViewerDownload Hi-res image Download (PPT)Characterization of GK-deficient of of GK in the pancreatic β-cell was in isolated islets from days mice. A rise in the intracellular concentration in the β-cell is a in insulin 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 they into two in of basal those with normal calcium levels ± n = about 20% of the and those with higher basal calcium levels than about which some of the In the the increase in by glucose was completely while the increase by or arginine was normal (Fig. However, in islets with higher basal calcium the increase in not only by glucose but also by was completely and that by arginine impaired (data not shown). The of GK-deficient islets may due to a direct of the impaired glucose metabolism in GK-deficient islets or may a of the and other in wild-type or heterozygous GK-deficient were islets with higher basal calcium levels than but its was than of GK-deficient increase in intracellular calcium concentration of islets by mM glucose, 10 or mM calcium levels in the wild-type, heterozygous, and homozygous islets are ± ± and ± respectively. are expressed in as mean ± S.E. (n = insulin secretion in response to the indicated secretagogues. are expressed in of insulin 10 as mean ± S.E. (n = the wild-type the and the homozygotes Insulin secretion in response to or 10 mM glucose from null islets was not p < 0.01; ∗, p < with the Large Image Figure ViewerDownload Hi-res image Download insulin secretion using the incubation method (Fig. Insulin secretion from heterozygous GK-deficient islets in response to 0.1 mM or mM glucose was but that in response to 10 mM glucose was significantly impaired with wild-type in insulin secretion in response to mM glucose was the other insulin secretion in response to or arginine was In homozygous GK-deficient was some basal insulin secretion at 0.1 mM glucose, presumably due to the activity of increase in insulin secretion in response to mM glucose was completely In insulin secretion in response to arginine was insulin secretion in response to it was by about on the method of (Fig. since the islets used were to a of those with normal and those with higher basal calcium levels to 20% and of the which were and to in calcium these results as suggesting that islets with normal basal calcium levels may have responded to in insulin insulin response to a physiological in glucose concentration was impaired in heterozygous GK-deficient islets and completely defective in homozygous GK-deficient islets the presence of the that GK serves as a glucose sensor molecule for insulin is consistent with the smaller increments in serum insulin levels after a glucose load in heterozygous mice (Fig. of increments in insulin levels in homozygous mice in spite of (Fig. and the in human with GK J. K. N. Stoffel M. Takeda J. Passa P. Cohen D. Bell G.I. Velho G. Froguel P. J. Clin. Invest. 1994; PubMed Scopus Google Scholar). GK-deficient islets responded to in insulin secretion normally to arginine and to some extent to that GK is not for insulin secretion in response to these secretagogues. It also that insulin secretion in response to was suggesting that GK may an role in insulin secretion in response to some of as in The heterozygous or homozygous mutant mice described provide the first animal model of diabetes with a defect in insulin and into the and development of human 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.