Glucose-dependent insulin release from genetically engineered K cells.

Genetic engineering of non-beta cells to release insulin upon feeding could be a therapeutic modality for patients with diabetes. A tumor-derived K-cell line was induced to produce human insulin by providing the cells with the human insulin gene linked to the 5'-regulatory region of the gene encoding glucose-dependent insulinotropic polypeptide (GIP). Mice expressing this transgene produced human insulin specifically in gut K cells. This insulin protected the mice from developing diabetes and maintained glucose tolerance after destruction of the native insulin-producing beta cells.

Cloning and gene structure of rat phosphatidylcholine transfer protein, Pctp.

Phosphatidylcholine transfer protein (PC-TP) is a cytosolic lipid transfer protein that promotes intermembrane transfer of phosphatidylcholines but no other phospholipids. Although its physiological function remains unknown, phosphatidylcholine transfer protein is enriched in liver and evidence from model systems suggests a role in hepatocellular selection and transport of biliary phospholipids. To facilitate in vivo studies, a cDNA encoding rat PC-TP was cloned by library screening and 5'-rapid amplification of cDNA ends. Genomic cloning demonstrated the rat Pctp gene spans 10. 8kb and is comprised of six exons. The putative transcription initiation site was identified 50bp upstream of the translation initiation site. Nucleotide sequence analysis of the 5'-flanking region revealed a CAAT- but no TATA-box. Transient transfection of a series of 5'-deleted Pctp-promoter-firefly luciferase constructs into Reuber H35 rat hepatoma cells, which express Pctp mRNA, and Gunn rat fibroblasts, which do not, suggest that cis-acting elements in a 637bp promoter region contribute to enhanced expression of PC-TP in liver.

Structure of the rat glucose-dependent insulinotropic polypeptide receptor gene.

The Glucose-dependent insulinotropic polypeptide receptor (GIPR) is a member of the secretin-vasoactive intestinal polypeptide family of G-protein coupled receptors possessing seven transmembrane domains. We report here the cloning and the exon-intron structure of the rat GIPR gene, along with the identification and characterization of its 5'-flanking region. The coding region of the GIPR gene spans approximately 10.2 kilobases and contains 13 exons. Three additional exons, two encoding either 5' or 3' untranslated sequences and one contained in a novel alternatively spliced mRNA, were identified. The 5'-flanking sequences contained a number of transcription factor binding motifs, including a cAMP response element, an octamer binding site, three SP1 sites and an initiator element. However, neither a CAAT motif nor TATA box were found. Transient transfection assays demonstrated that the 5'-flanking region of the GIPR gene can efficiently promote transcription in RIN38 cells and that deletion of 50 base pairs containing a potential SPI binding sites leads to a 2.4-fold loss of transcriptional activity. In addition, transient transfection experiments comparing the relative promoter activities of 5'-flanking sequences of the GIPR gene in RIN38 and rat-2 cells suggests that distal negative regulatory sequences may control cell-specific expression.

Functional GIP receptors are present on adipocytes.

In addition to its important role in maintaining glucose homeostasis, it has recently become apparent that glucose-dependent insulinotropic polypeptide (GIP) is also involved in different steps of lipid metabolism. GIP has been shown to stimulate the release of lipoprotein lipase from fat, as well as increase the rate of fat incorporation into adipose tissue. Moreover, GIP has been shown to increase the clearance rate of chylomicrons in the circulation and to inhibit the action of glucagon. Despite evidence for GIP effects on fat tissue, GIP receptors have not been identified in fat cells or tissues. The present study was undertaken to identify GIP receptors in isolated adipocytes, as well as to identify GIP receptors in the established fat cell line, differentiated 3T3-L1. RNAse protection analysis demonstrated the presence of GIP receptor transcripts in rat adipocytes. A polyclonal GIP receptor antiserum directed at the N-terminus of the receptor detected the presence of GIP receptors in both rat fat and differentiated 3T3-L1 cells by Western blot analysis. Moreover, [125I] GIP binding assays revealed both specific and displaceable GIP binding sites in differentiated 3T3-L1 cells (IC50 = 10(-9) M). When undifferentiated 3T3-L1 cells, which appear to express relatively few GIP receptors, were incubated in the presence of GIP, no effect on intracellular cAMP accumulation was detected. In contrast, the inclusion of 10 nM GIP in the incubation medium increased cAMP accumulation in rat fat cells and differentiated 3T3-L1 cells. This increase in cAMP accumulation was abolished with the specific GIP receptor antagonist GIP(7-30)NH2. The results of these studies indicate that GIP receptors are present in fat cells and are up-regulated when 3T3-L1 cells undergo differentiation to become adipocytes. Furthermore, the increase in intracellular cAMP accumulation detected upon ligand binding indicates that these receptors are functional.

Cell-specific expression of the glucose-dependent insulinotropic polypeptide gene in a mouse neuroendocrine tumor cell line.

Glucose-dependent insulinotropic polypeptide (GIP) is a 42-amino acid gastrointestinal regulatory peptide that, in the presence of glucose, stimulates insulin secretion. GIP is expressed in K cells of the small intestine and in cells of the submandibular salivary gland. Using a rat GIP cDNA as a specific probe, we screened a number of established cell lines for the expression of GIP mRNA. STC-1 cells, a cell line derived from a mouse neuroendocrine tumor, were found to express high levels of GIP mRNA. GIP-specific transcripts were not detected in other cell lines tested, which included cells of intestinal, salivary, and endocrine origin. Analysis of GIP-luciferase fusions identified two promoters, a distal and a proximal promoter, upstream of the translation initiation codon for GIP. The distal promoter, located upstream of position +1, corresponds to the principal promoter of the GIP gene and can promote cell-specific transcription. Sequential deletion and site-directed mutational analysis of the distal promoter demonstrated that the sequence between -193 and -182 determines cell-specific expression of GIP. Contained in this region is a consensus GATA motif, suggesting that a member of the GATA family of DNA-binding proteins is involved in the cell-specific regulation of the GIP gene.

Activation of tumor suppressor genes in nontumorigenic revertants of the HeLa cervical carcinoma cell line.

Two nontransformed revertants of HeLa cells, designated HA and HF, were isolated using a selection procedure based on prolonged retention of the fluorescent dye rhodamine 123 within the mitochondria of HeLa (ATCC CCL2) cells versus normal epithelial cells. Unlike the parental HeLa cells, the revertants expressed markedly reduced levels of the bone-liver-kidney, placental, and intestinal isoforms of alkaline phosphatase, exhibited a flat nonrefractile morphology, and failed to grow in suspension culture. The revertant clones had > 100-fold reduced cloning efficiencies in semisolid medium relative to HeLa cells and failed to induce s.c. tumors when injected into nude mice. Both revertant clones have retained nontransformed phenotypes after 5 years of continuous culture. Southern blot analyses performed with human papillomavirus 18-specific DNA probes indicated that the integrated viral sequences present in HeLa cells remained intact in the revertants. Furthermore, the level of the polycistronic mRNAs encoding the viral E6 and E7 oncogenes were comparable in the parental HeLa cell line and the revertants. Western blot analyses of immunoprecipitated human papillomavirus 18 E6 and E7 proteins further demonstrated that the levels of these viral oncoproteins were comparable in HeLa cells and revertants. Infection with helper-free, defective retroviruses that express E6, E7 or E6 and E7 oncogenes failed to retransform the revertants, suggesting that their nontransformed phenotype did not result from mutations in these viral oncogenes. Cell fusion experiments indicated that the revertant phenotypes of HA and HF cells resulted from mutations in cellular genes that activate one or more tumor suppressor genes.

Chronic desensitization of the glucose-dependent insulinotropic polypeptide receptor in diabetic rats.

Rats were rendered diabetic by streptozotocin, after which serum glucose-dependent insulinotropic polypeptide (GIP) levels, duodenal mucosal GIP content, and GIP mRNA levels were nine times, 50% and 80%, respectively, greater than in control rats. To determine whether an increase in GIP gene expression might induce chronic desensitization of its receptor, normal rats were subjected to continuous intravenous GIP infusion. Serum GIP levels increased gradually in GIP-infused rats, and by 4 h a threefold increase was detected. In response to GIP infusion, the serum insulin concentration increased at 30 min, followed by a gradual decrease, and at 4 h, no increase in insulin levels was detected despite a sustained elevated serum GIP level. The response to glucagon-like peptide-1 (GLP-1) was preserved, a reporter cell line (LGIPR2) stably transfected with rat GIP receptor cDNA was studied. GIP stimulated adenosine 3', 5'-cyclic monophosphate (cAMP) production in LGIPR2 cells, which was first detected after 1 h of stimulation, reached maximum level at 4 h, and returned to basal concentrations by 16 h. Additional stimulation with GIP at 16 h did not affect cAMP generation, indicating desensitization of the GIP receptor by the ligand. In contrast, a response to prostaglandin E1 or forskolin in GIP-desensitization was a receptor-specific process. The results of these studies indicate that GIP gene expression is enhanced in diabetic animals and that elevated serum GIP level induces chronic desensitization of the GIP receptor in vivo and in a stably transfected cell line.

Glucose-dependent insulinotropic peptide (GIP) gene expression in the rat salivary gland.

Previous studies have indicated that following nutrient ingestion, GIP is released principally from the upper small intestine. In addition to its presence in the rat small intestine, GIP transcripts have also been localized to the submandibular salivary gland (SSG). The present studies were directed to further characterize expression of the GIP gene in the SSG. Pregnant rats were sacrificed at gestational days 18 and 20, followed by the removal of rat fetuses. The duodenum pancreas, and SSG were then excised from the fetuses, as well as from neonatal pups at ages 1, 3, 7, 10, 14, and 21 days. RNA was extracted and measured by Northern blot analysis using specific rat GIP probes. GIP transcripts were first detected in the duodenum in the 18-day fetus and reached maximum levels at birth. In contrast, GIP mRNA was not observed in the SSG until 10 days postnatally and was not detected at all in either the fetal or neonatal pancreas. In situ hybridization of the SSG using an 35S-labelled antisense GIP RNA probe demonstrated expression of the GIP gene to be limited to ductal cells, with no transcripts present in acini. In separate experiments, rats fasted overnight were given water or 10% glucose. While no changes were detected in water-fed rats following oral glucose ingestion, small, but significant increases in SSG GIP gene expression were detected at 60 and 240 min. The results of these initial studies suggest the possibility of a functional role for GIP in the rat salivary gland by the demonstration of GIP mRNA in the SSG by Northern analysis and in situ hybridization, as well as by an increase in SSG GIP gene expression following a glucose meal.

Transformation effector and suppressor genes.

Much has been learned about the molecular basis of cancer from the study of the dominantly acting viral and cellular oncogenes and their normal progenitors, the proto-oncogenes. More recent studies have resulted in the isolation and characterization of several genes prototypic of a second class of cancer genes. Whereas oncogenes act to promote the growth of cells, members of this latter class of genes act to inhibit cellular growth and are believed to contribute to the tumorigenic phenotype only when their activities are absent. This new class of cancer genes is referred to by a number of different names including; anti-oncogenes, recessive oncogenes, growth suppressor genes, tumor suppressor genes and emerogenes. Although only a few of these cancer genes have been identified, to date, it is likely that many additional genes of this class await identification. A third class of genes, necessary for the development of the cancer phenotype, is comprised of the transformation effector genes. These are normal cellular genes that encode proteins that cooperate with or activate oncogene functions and thereby induce the development of the neoplastic phenotype. The inactivation of transformation effector functions would therefore inhibit the ability of certain dominantly acting oncogenes to transform cells. The approaches outlined here describe functional assays for the isolation and molecular characterization of transformation effector and suppressor genes.

Functional in vitro assays for the isolation of cell transformation effector and suppressor genes.

Malignant transformation may be viewed as an imbalance between signals inducing cell growth and signals leading to growth inhibition, differentiation, or senescence. A basic understanding of how these counterbalancing forces interact to regulate normal cell growth is the prerequisite to comprehending the mechanisms of tumorigenesis. Identification and characterization of the gene products implicated in these regulatory pathways is the first step toward understanding the disease process. The studies outlined here provide the potential basis for isolating and molecularly characterizing transformation effector and suppressor genes, which must respectively function in the positive and negative regulation of normal cell growth. The general strategy used involves the isolation and molecular characterization of nontransformed variants (revertants) from populations of tumor cells. The selection of revertants is facilitated by the ability to separate normal from transformed cells by fluorescence-activated sorting. The basis for this separation is the differential retention of the fluorescent dye rhodamine 123 in the mitochondria of normal versus transformed cells. Using this approach, we have isolated revertants from a mutagenized population of v-fos-transformed Rat-1 fibroblasts. Characterization of these clones indicated that they had sustained causal mutations in transformation effector genes. The unmutated effector genes are being identified and molecularly cloned by isolating retransformed clones from revertant cell lines that have been transfected with DNA or cDNA from normal primary cells. The same selection protocol has also been used to isolate revertants from tumor cell lines that have been transfected with DNA or cDNA from primary cells. The putative tumor-suppressor genes present in these revertants are currently being analyzed.

Construction of a fused LuxAB gene by site-directed mutagenesis.

Bacterial luciferases are heteropolymetric enzymes consisting of two non-identical subunits (alpha and beta). The two polypeptides are produced by transcription in the same direction of two genes, luxA and luxB, located immediately adjacent to each other and separated by only 29 base pairs in the Vibrio harveyi genome. Using site-directed mutagenesis, stop codons after luxA were eliminated and the luxB gene was placed in-frame with luxA, resulting in a fused luxAB gene. Transcription of two luxAB mutant genes from the bacteriophage T7 promoter and translation in Escherichia coli resulted in the synthesis of fused polypeptides containing the alpha and the beta subunits of luciferase linked by either a single amino acid residue or a decapeptide. E. coli synthesizing the latter fusion protein with the decapeptide linker expressed a level of luminescence comparable to E. coli containing the wild type genes while E. coli synthesizing the polypeptide with a single amino acid as a linker expressed about 2000-fold lower light. These results provide the basis for generating a bacterial luciferase system that can be expressed under the control of a single promoter in both eukaryotic and prokaryotic systems.