Bryant P. Bullock

The incretin hormone glucagon-like peptide-1 (GLP-1) is an important regulator of postprandial insulin secretion. In addition to its insulinotropic actions on pancreatic beta-cells, GLP-1 enhances glucose disposal by insulin-independent mechanisms, suggesting that GLP-1 receptors are located on extrapancreatic tissues. In this study, we examined the tissue distribution of GLP-1 receptor (GLP-lR) messenger RNA (mRNA) in rat by RNAse protection, RT-PCR, and in situ hybridization. We identified GLP-1R mRNA in the lung, pancreatic islets, stomach, and kidney by the RNAse protection assay. RT-PCR analysis also detected GLP-1R mRNA in the hypothalamus and heart. In situ hybridization experiments identified receptor mRNA in the gastric pits of the stomach, large nucleated cells in the lung, crypts of the duodenum, and pancreatic islets. No localized specific grains were found in kidney, skeletal muscle, heart, liver, or adipocytes. These results indicate that sequences corresponding to the cloned rat islet GLP-1 receptor are expressed in the pancreatic islets, lung, hypothalamus, stomach, heart, and kidney but not in adipose, liver, and skeletal muscle. Further, the GLP-1 receptor expressed in the kidney and heart may be structural variants of the known receptor. Therefore, the observed extrapancreatic actions of GLP-1 may not be strictly confined to interactions with the defined GLP-1 receptor.

Aldehyde reductase [EC 1.1.1.2] and aldose reductase [EC 1.1.1.21] are monomeric NADPH-dependent oxidoreductases having wide substrate specificities for carbonyl compounds. These enzymes are implicated in the development of diabetic complications by catalyzing the reduction of glucose to sorbitol. Enzyme inhibition as a direct pharmacokinetic approach to the prevention of diabetic complications resulting from the hyperglycemia of diabetes has not been effective because of nonspecificity of the inhibitors and some appreciable side effects. To understand the structural and evolutionary relationship of these enzymes, we cloned and sequenced cDNAs coding for aldose and aldehyde reductases from human liver and placental cDNA libraries. Human placental aldose reductase (open reading frame of 316 amino acids) has a 65% identity (identical plus conservative substitutions) to human liver and placental aldehyde reductase (open reading frame of 325 amino acids). The two sequences have significant identity to 2,5-diketogluconic acid reductase from corynebacterium, frog rho-crystallin, and bovine lung prostaglandin F synthase (reductase). Southern hybridization analysis of human genomic DNA indicates a multigene system for aldose reductase, suggesting the existence of additional proteins. Thus, the aldo-keto reductase superfamily of proteins may have a more significant and hitherto not fully appreciated role in general cellular metabolism.

Recombinant DNA clones encoding the neurotensin/neuromedin N precursor protein have been isolated from both bovine hypothalamus cDNA and rat genomic libraries using a heterologous canine cDNA probe. Nucleotide sequence analysis of these clones and comparison with the previously determined canine sequence has revealed that 76% of the amino acid residues are conserved in all three species. The protein precursor sequences predicted from bovine hypothalamus and canine intestine cDNA clones vary at only 9 of 170 amino acid residues suggesting that within a species identical precursors are synthesized in both the central nervous system and intestine. The rat gene spans approximately 10.2 kilobases (kb) and is divided into four exons by three introns. The neurotensin and neuromedin N coding domains are tandemly positioned on exon 4. RNA blot analysis has revealed that the rat gene is transcribed to yield two distinct mRNAs, 1.0 and 1.5 kb in size, in all gastrointestinal and all neural tissues examined except the cerebellum. There is a striking variation in the relative levels of these two mRNAs between brain and intestine. The smaller 1.0-kb mRNA greatly predominates in intestine while both mRNA species are nearly equally abundant in hypothalamus, brain stem, and cortex. Sequence comparisons and RNA blot analysis indicate that these two mRNAs result from the differential utilization of two consensus poly(A) addition signals and differ in the extent of their 3' untranslated regions. The relative combined levels of the mRNAs in various brain and intestine regions correspond roughly with the relative levels of immunologically detectable neurotensin except in the cerebral cortex where mRNA levels are 6 times higher than anticipated.

The cAMP response element binding protein CREB activates the transcription of genes in response to phosphorylation by cAMP-dependent protein kinase A (PKA) and other protein kinases. Phosphorylated CREB activates transcription by recruiting transcriptional co-activators such as the CREB binding protein. Here, we describe experiments that analyze the effects of phosphorylation on the DNA binding affinity of CREB and the structural characteristics of the CREB/DNA complex in solution. Analysis of deletion mutants of CREB indicate that amino acid sequences within the transactivation domain promote high-affinity binding of CREB to fluorescently labeled oligonucleotides containing cAMP response elements. In vitro experiments indicate that phosphorylation is processive between PKA as the initial kinase and glycogen synthase kinase-3 (GSK-3) but not casein kinase II as the secondary kinase. Fluorescent electrophoretic mobility shift assays show that phosphorylation by PKA results in a 3-5-fold increase in the binding affinity of CREB to both the symmetrical somatostatin CRE (SMS-CRE) and the asymmetric somatostatin upstream element (SMS-UE). Processive phosphorylation of CREB by GSK-3 attenuates the enhanced DNA binding in response to PKA thus acts as an inhibitor of PKA-induced binding. Ferguson plot analyses demonstrate that phosphorylation of CREB by PKA and GSK-3 result in an increase in the spherical size and the net positive surface charge of the CREB/DNA complex. Moreover, these analyses uncovered the unexpected finding that CREB associates as a tetramer both in the presence and absence of DNA. These findings suggest a model by which phosphorylation of CREB alters the secondary structure and charge characteristics of the CREB/DNA complex resulting in an alteration in binding affinity.