Pancreatic β-Cell-specific Targeted Disruption of Glucokinase Gene

Abstract

Mice carrying a null mutation in the glucokinase (GK) gene in pancreatic ␤-cells, but not in the liver, were generated by disrupting the ␤-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 noninsulin-dependent diabetes mellitus.Glucokinase (GK), 1 mainly expressed in pancreatic ␤-cells and the liver, is thought to constitute a rate-limiting step in glucose metabolism in these tissues (1-4). Since insulin secretion parallels glucose metabolism and the high K m 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 ␤-cell (1, 5). 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 -8). However, since all the mutations in humans so far occur in the region of the gene that is common to pancreatic ␤-cells and hepatocytes (9), and are heterozygous, it may not have been possible to fully reveal physiological roles of pancreatic ␤-cell GK either in vivo or in vitro. To this end, mice carrying a null mutation in the GK gene in pancreatic ␤-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 Experiments-A DNA fragment including the pancreatic ␤-cell-specific exon 1␤ of the GK gene was cloned from a BALB/c mouse genomic library (Clontech). A BamHI site was introduced 30 base pairs 3Ј to the translation initiation codon of GK by the Kunkel method (10). A neomycin resistance gene (neo r ) with a pgk-1 promoter but without a poly(A) ϩ addition signal was substituted for the XbaI-BamHI fragment in the exon 1␤. A diphtheria toxin A fragment gene (DTA) with a MC1 promoter was lig...

Role of the GLUT 2 Glucose Transporter in the Response of the L-type Pyruvate Kinase Gene to Glucose in Liver-derived Cells

“…However, glucokinase could be replaced by other insulin-independent hexokinases, in vivo and ex vivo. In vivo, knocked out mice specifically deficient in glucokinase in liver have an almost normal glucose regulation (32), whereas deficiency in pancreas glucokinase leads to neonatal death from severe insulin-dependent diabetes mellitus (33). Ex vivo, we have shown that hepatoma cells with hexokinase 1 (2, and this paper) respond to glucose in modulating transcription of glucose-responsive genes, e.g.…”

“…In endocrine pancreas ␤ cells, the predominant glucose-sensor has been definitively identified as glucokinase by knock out of the gene in mice (32,33), and the significance of the specific GLUT 2 expression in rodent ␤ cells is still disputed, especially as the amount of the GLUT 2 isoform seems to be very low in human ␤ cells (34,35). German (36) has shown that ␤ cells overexpressing GLUT 1 do not lose their ability to sense changes in glucose concentration within the physiological range and to respond by an appropriate stimulation of the insulin gene promoter.…”

Insulin effect during embryogenesis determines fetal growth: a possible molecular link between birth weight and susceptibility to type 2 diabetes.

“…In addition, because human G c k + / -fetuses had decreased Gck activity in the liver, the possibility that the resultant impaired hepatic glycogen accumulation (12) may lead to alterations in fetal development cannot be excluded. In this regard, we were able to eliminate selectively the expression of the -cell isoform of Gck without affecting the expression of the liver isoform in mice by disrupting the first exon specific to the -cell type Gck (13). To understand the relationship between birth weight and fetal insulin secretion, we measured birth weight and insulin levels of neonates derived from crosses of male pancreatic -cell type Gck knockout (G c k + / -) mice (13) and female wildtype (WT) or G c k + / -m i c e .…”

“…In this regard, we were able to eliminate selectively the expression of the -cell isoform of Gck without affecting the expression of the liver isoform in mice by disrupting the first exon specific to the -cell type Gck (13). To understand the relationship between birth weight and fetal insulin secretion, we measured birth weight and insulin levels of neonates derived from crosses of male pancreatic -cell type Gck knockout (G c k + / -) mice (13) and female wildtype (WT) or G c k + / -m i c e . Low birth weight can also be associated with insulin resistance resulting from genetic defects that influence insulin action during embryogenesis.…”

Upregulation of β-cell genes and improved function in rodent islets following chronic glucokinase activation

“…The pancreatic GK isoform is an absolute requirement for survival, as gk del/del (either global or b-cell specific) is lethal, whereas global or b-cell-specific heterozygous (gk del/wt ) deletion causes only modest hyperglycaemia (Bali et al 1995, Grupe et al 1995, Terauchi et al 1995. Deletion of liver GK leads to mild hyperglycaemia but decreases glucose utilisation and glycogen synthesis (Postic et al 1999).…”