A pancreatic islet-specific glucose-6-phosphatase-related protein (IGRP) was cloned using a subtractive cDNA expression cloning procedure from mouse insulinoma tissue. Two alternatively spliced variants that differed by the presence or absence of a 118-bp exon (exon IV) were detected in normal balb/c mice, diabetic ob/ob mice, and insulinoma tissue. The longer, 1901-bp full-length cDNA encoded a 355-amino acid protein (molecular weight 40,684) structurally related (50% overall identity) to the liver glucose-6-phosphatase and exhibited similar predicted transmembrane topology, conservation of catalytically important residues, and the presence of an endoplasmic reticulum retention signal. The shorter transcript encoded two possible open reading frames (ORFs), neither of which possessed His174, a residue thought to be the phosphoryl acceptor (Pan CJ, Lei KJ, Annabi B, Hemrika W, Chou JY: Transmembrane topology of glucose-6-phosphatase. J Biol Chem 273:6144-6148, 1998). Northern blot and reverse transcription-polymerase chain reaction analysis showed that the mRNA was highly expressed in pancreatic islets and expressed more in beta-cell lines than in an alpha-cell line. It was notably absent in tissues and cell lines of non-islet neuroendocrine origin, and no other major tissue source of the mRNA was found. During development, it was expressed in parallel with insulin mRNA. The mRNA was efficiently translated and glycosylated in an in vitro translation/membrane translocation system and readily transcribed into COS 1, HIT, and CHO cells using cytomegalovirus or Rous sarcoma virus promoters. Whereas the liver glucose-6-phosphatase showed activity in these transfection systems, the IGRP failed to show glucose phosphotransferase or phosphatase activity with p-nitrophenol phosphate, inorganic pyrophosphate, or a range of sugar phosphates hydrolyzed by the liver enzyme. While the metabolic function of the enzyme is not resolved, its remarkable tissue-specific expression warrants further investigation, as does its transcriptional regulation in conditions where glucose responsiveness of the pancreatic islet is altered.

Glucose-6-phosphatase (G6Pase) catalyzes the final step in the gluconeogenic and glycogenolytic pathways, the hydrolysis of glucose-6-phosphate (G6P) to glucose and phosphate. This paper describes the identification and characterization of a human cDNA and gene encoding a ubiquitously expressed G6Pase catalytic subunit-related protein (UGRP). The ORF of this UGRP cDNA encodes a protein (346 amino acids (aa); M r 38 709) which shares 36% overall identity to the human G6Pase catalytic subunit (357 aa; M r 40 487). UGRP exhibits a similar predicted transmembrane topology and conservation of many of the catalytically important residues with the G6Pase catalytic subunit; however, unlike the G6Pase catalytic subunit, UGRP does not catalyze G6P hydrolysis. UGRP mRNA was detected by RNA blot analysis in every tissue examined with the highest expression in muscle. Database analysis showed that the human UGRP gene is composed of six exons, spans ∼5·4 kbp of genomic DNA and is located on chromosome 17q21 with the G6Pase catalytic subunit gene. The UGRP gene transcription start sites were mapped by primer extension analysis, and the activity of the UGRP gene promoter was analyzed using luciferase fusion gene constructs. In contrast to the G6Pase catalytic subunit gene promoter, the UGRP promoter was highly active in all cell lines examined.

Changes in extracellular pH affected insulin output from pancreatic islets stimulated with either glucose or alpha-ketoisocaproate. The extracellular pH at which the highest secretory response occurred was not identical in all cases, being shifted to alkaline values as the concentration of the nutrient secretagogue was increased. The output of insulin correlated with the nutrient-induced increment in 45Ca net uptake, but not with the rate of nutrient oxidation. By reference to changes in intracellular pH observed in islets exposed to either acidic media or increasing concentrations of alpha-ketoisocaproate, and taking into account a titration curve for the buffering capacity of islet homogenates, the optimal intracellular pH for insulin release was found to approximate a value of 0.09 pH unit below basal pH. It is postulated that, in the process of nutrient-induced insulin release, modest changes in intracellular pH participate in the multifactorial coupling between metabolic, ionic and secretory events.

Glucose-6-phosphatase (G6Pase) catalyzes the final step in the gluconeogenic and glycogenolytic pathways, the hydrolysis of glucose-6-phosphate (G6P) to glucose and phosphate. This paper describes the identification and characterization of a cDNA and the gene encoding the mouse ubiquitously expressed G6Pase catalytic subunit-related protein (UGRP). The open reading frame of this UGRP cDNA encodes a protein (346 amino acids (aa); M r 38 755) that shares 36% overall identity (56% similarity) with the mouse G6Pase catalytic subunit (357 aa; M r 40 454). UGRP exhibits a similar predicted transmembrane topology and conservation of many of the catalytically important residues with the G6Pase catalytic subunit; however, unlike the G6Pase catalytic subunit, UGRP does not catalyze G6P hydrolysis and does not contain a carboxy-terminal di-lysine endoplasmic reticulum retention signal. UGRP mRNA was detected by RNA blot analysis in every mouse tissue examined with the highest expression in heart, brain, testis and kidney. Database analysis showed that the mouse UGRP gene is composed of six exons, spans ∼4·2 kbp of genomic DNA and is located on chromosome 11 along with the G6Pase catalytic subunit gene. The UGRP gene transcription start sites were mapped by primer extension analysis, and the activity of the mouse UGRP gene promoter was analyzed using luciferase fusion gene constructs. In contrast to the G6Pase catalytic subunit gene promoter, the UGRP promoter was highly active in all cell lines examined.

Summary. The capacities of rat lens and retina to accumulate sorbitol pathway intemiediates in streptozotocin induced diabetes were inve.stigated. The experiments performed were of two types:(i) investigations of the time course of the appearance of the pathway intermediates in lens and retina following the induction of diabetes, and (ii) investigation of the relationship of scirbitol pathway intermediate accumulations to varying doses of .strt'ptozotocin. The experimental objectives were to investigate the factors which control the activity of the pathway in vivo, and to a.ssfss the proposed relation.ship between sorbitol metabolism in the retina and the pathogenesis of diabetic small vessel disease in this tissue.The sorbitol which accumulated in the retina of the diabetic animal (approximately 1-5 iimolc/g protein) was formed by the intrinsic metabolic activity of the tissne and was not attributed to transport from an external metabolic site. The accunmlation of this intermediate was directly related to the plasma gincose concentration and could not be related to other parameters of the diabetic state, e.g. plasma free fatty acid concentration, ketonaemia.It was confirmed that the rat lens accumulated sorbitol (approximately 80 (imole/g protein) and fructose (approximately 20 n'Tiole/g protein) in diabetes, and that such accumulations were related to the glucose concentration of the aqueous humour. The possibility that factors associated with kctoacidosis aflected the relati\e concentration.s of lens sorbitol and fructose was suggested by the results.An hypothesis which invoked sorbitol iiccuninlation to osmotically signifieant concentrations within the retina as a pathogenic agent in diabetic small ve.ssel disease in this tissue conld not be directly supported by the results. Alternate mechanisms by which low concentrations of sorbitol iu diabetic retina could act as a pathogenic stimulus are discussed.