Glucokinase (GK) gene mutations cause diabetes mellitus in both humans and mouse models, but the pathophysiological basis is only partially defined. We have used cre-loxP technology in combination with gene targeting to perform global, ␤ cell-, and hepatocyte-specific gene knock-outs of this enzyme in mice. Gene targeting was used to create a triple-loxed gk allele, which was converted by partial or total Cre-mediated recombination to a conditional allele lacking neomycin resistance, or to a null allele, respectively. ␤ cell-and hepatocytespecific expression of Cre was achieved using transgenes that contain either insulin or albumin promoter/ enhancer sequences. By intercrossing the transgenic mice that express Cre in a cell-specific manner with mice containing a conditional gk allele, we obtained animals with either a ␤ cell or hepatocyte-specific knock-out of GK. Animals either globally deficient in GK, or lacking GK just in ␤ cells, die within a few days of birth from severe diabetes. Mice that are heterozygous null for GK, either globally or just in the ␤ cell, survive but are moderately hyperglycemic. Mice that lack GK only in the liver are only mildly hyperglycemic but display pronounced defects in both glycogen synthesis and glucose turnover rates during a hyperglycemic clamp. Interestingly, hepatic GK knock-out mice also have impaired insulin secretion in response to glucose. These studies indicate that deficiencies in both ␤ cell and hepatic GK contribute to the hyperglycemia of MODY-2.

Glucokinase (GK) gene transcription initiates in the islet (␤-cell), gut, and brain from promoter sequences residing ϳ35 kbp upstream from those used in liver. Expression of ␤GK is controlled in ␤-cells by cellenriched (i.e. pancreatic duodenal homeobox 1 [PDX-1]) and ubiquitously (i.e., Pal) distributed factors that bind to and activate from conserved sequence motifs within the upstream promoter region (termed ␤GK). Here, we show that a conserved E-box element also contributes to control in the islet and gut. ␤GK promoter-driven reporter gene activity was diminished by mutating the specific sequences involved in E-boxmediated basic helix-loop-helix factor activator binding in islet ␤-cells and enteroendocrine cells. Gel shift assays demonstrated that the ␤GK and insulin gene E-box elements formed the same cell-enriched (BETA2: E47) and generally distributed (upstream stimulatory factor [USF]) protein-DNA complexes. ␤GK E-boxdriven activity was stimulated in cotransfection assays performed in baby hamster kidney (BHK) cells with BETA2 and E47, but not USF. Chromatin immunoprecipitation assays performed with BETA2 antisera showed that BETA2 occupies the upstream promoter region of the endogenous ␤GK gene in ␤-cells. We propose that BETA2 (also termed NeuroD1) regulates ␤GK promoter activity. Diabetes 52: [403][404][405][406][407][408] 2003

Congenital absence of the pancreas is an extremely rare condition. We participated in the care of a patient with an unusual presentation of neonatal diabetes attributable to agenesis of the pancreas. Additional clinical features of the patient included cardiac septal defects, gall bladder agenesis and duodenal malrotation. Appropriate institution of insulin, exocrine pancreatic supplements and surgical repair of the cardiac and intestinal anomalies resulted in the infant's survival. Of the reported cases of congenital pancreas agenesis, two cases have been ascribed to mutations in the insulin promoter factor-1(Ipf-1) gene. Deletion of the Ipf-1-homolog pdx-1 in mice results in the failure of pancreas to develop. Analysis of both exons of the Ipf-1 coding sequence from the presented patient's genomic DNA, however, did not identify a mutation. These results suggest that a congenital or genetic perturbation occurred in this infant most likely before the appearance of dorsal pancreatic bud in the 3 mm long embryonic stage, around the embryonic day 25 in human development, before the onset of Ipf-1 expression.

The role of the exercise-induced increment in epinephrine was studied in five adrenalectomized (ADX) and in six normal dogs (C). Experiments consisted of an 80-min equilibration period, a 40-min basal period, and a 150-min exercise period. ADX were studied with epinephrine replaced to basal levels during rest and to increased levels during exercise to simulate its normal rise (HE) and on a separate day with epinephrine maintained at basal levels throughout the study (BE). Cortisol was replaced during rest and exercise in ADX so as to simulate the levels seen in C. Glucose was infused as needed in ADX to maintain the glycemia evident during exercise in C. Glucose production (Ra) and utilization (Rd) were assessed isotopically. In C, epinephrine had risen by 95 +/- 25 pg/ml by the end of exercise. In HE, the increment in epinephrine (117 +/- 29 pg/ml) was similar to that seen in C, whereas in BE epinephrine fell by 18 +/- 9 pg/ml. Basal norepinephrine levels were 139 +/- 9, 260 +/- 25, and 313 +/- 33 pg/ml in C, HE, and BE, respectively. In response to exercise, norepinephrine increased by nearly twofold in all protocols. Basal and exercise-induced changes in plasma glucagon and insulin were similar in C and ADX. Ra increased similarly in C (5.3 +/- 0.6 mg.kg-1.min-1) and HE (4.9 +/- 0.6 mg.kg-1.min-1). In BE, Ra rose normally for the initial 90 min but then declined resulting in a rise of only 2.9 +/- 0.5 mg.kg-1.min-1 after 150 min of exercise.(ABSTRACT TRUNCATED AT 250 WORDS)

Aims/hypothesis. The upstream glucokinase (βGK) promoter has previously been shown to contain two 9-bp sequences, termed the Pal motifs, that are conserved in humans, rats and mice. These motifs are necessary for expression of the βGK promoter in pancreatic beta cell lines and pituitary corticotrope cell lines. DNA probes containing the Pal motifs bind celltype-specific protein complexes, but these motifs have not been completely characterised. Methods. Methylation interference and ultravioletcrosslinking analysis were utilised to characterise, at the single nucleotide level, sites of protein binding within the elements themselves. To determine the function of these elements, mutational analysis of the βGK promoter and of multimerised GK promoter sequences was performed.Results. Both Pal elements are 14 bp in length and have dyad symmetry. However, while the Pal-1 element is a perfect inverted repeat (GTCACCA-TGGT-GAC), the Pal-2 element (GTCACCA-TAGAAAC) is an imperfect repeat. Ultraviolet-crosslinking analysis using nuclear extracts prepared from hamster insulinoma tumour (HIT) cells revealed that the three resolvable complexes that bind to the Pal-1 and Pal-2 elements contain different ratios of three proteins of different size (~90, 110 and 150 M r ). Mutation of a single nucleotide binding site abrogates βGK promoter activity. Multimerised repeats of the Pal-1 element augment transcription in HIT cells, but not in baby hamster kidney (BHK) cells. Conclusions/interpretation. These results suggest that different combinations of three proteins of different size bind to the Pal elements, probably as homodimers and heterodimers. Together, these results indicate that the βGK promoter contains two novel 14-bp elements that, when multimerised, exhibit enhancer activity specific to neuroendocrine cells.