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T L Andreone

Publications and source records attributed to T L Andreone.

9 recordsLinked to original sources
PubMed1989

The amino acid sequence of rat liver glucokinase deduced from cloned cDNA.

Rat liver glucokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) was purified to homogeneity, cleaved, and subjected to amino acid sequence analysis. Forty-five percent of the protein sequence was obtained, and this information was used to design oligonucleotide probes to screen a rat liver cDNA library. A 1601-base pair cDNA (GK1) contained an open reading frame that encoded the amino acid sequences found in the peptides used to generate the oligonucleotide probes. A second cDNA was subsequently identified (GK.Z2), which is 2346 base pairs long and corresponds to nearly the entire glucokinase mRNA. Blot transfer analysis of hepatic RNA showed that glucokinase mRNA exists as a single species of about 2400 nucleotides. Four hours of insulin treatment of diabetic rats resulted in a 30-fold induction of this mRNA. GK.Z2 has a long open reading frame which, with the known partial peptide sequence, allowed us to deduce the primary structure of glucokinase. The enzyme is composed of 465 amino acids and has a mass of 51,924 daltons. Glucokinase has 53 and 33% amino acid sequence identities with the carboxyl-terminal domains of rat brain hexokinase I and yeast hexokinase, respectively. If conservative amino acid replacements are also considered, glucokinase is similar to these two enzymes at 75 and 63% of positions, respectively. The putative glucose- and ATP-binding domains of glucokinase were identified, and these regions appear to be highly conserved in the hexokinase family of enzymes.

Amino Acid Sequence↗
PubMed1989

Rat glucokinase gene: structure and regulation by insulin.

The glucokinase gene is 15.5-kilobases long, appears to be present as a single copy, and contains 10 exons that range in size from 96 to 977 base pairs. The transcription start site was located 127 nucleotides upstream from the translation initiation codon. The 5' flanking DNA contains several regions similar to defined promoter elements. These include a probable "TATA box," an Sp1 binding site, and several elements related to liver-specific gene expression. In addition, we determined that transcription of the glucokinase gene increased at least 20-fold when diabetic rats were treated with insulin for 2 hr.

Animals↗
PubMed1984

Multihormonal regulation of phosphoenolpyruvate carboxykinase gene transcription. The dominant role of insulin.

We used a nuclear RNA transcript elongation assay to show that cAMP analogs and dexamethasone cause a selective increase of transcription of the P-enolpyruvate carboxykinase gene in H4IIE hepatoma cells. 8-(4-chlorophenylthio)-cAMP increased transcription within 5 min and the maximal rate, generally 10-15-fold above the basal rate, was attained by 30 min. This increase was of sufficient magnitude to account for the effect on mRNAPEPCK (for example, where PEPCK is phosphoenolpyruvate carboxykinase) accumulation. After the initial increase, and with continued presence of cAMP, transcription of this gene declined to a new steady-state level which was 2-3 times the basal value. The effect of cAMP analogs on P-enolpyruvate carboxykinase gene transcription was obtained in the absence of protein synthesis. This, and the rapidity of the response, indicates that the effect of cAMP is exerted directly on the P-enolpyruvate carboxykinase gene. Dexamethasone results in a specific, 6-fold increase of transcription, sufficient to account for the increase of mRNAPEPCK which follows treatment of H4IIE cells with this glucocorticoid. When 1 nM insulin was added to either untreated H4IIE cells, or cells first treated with a cAMP analog or dexamethasone, there was a marked reduction of cytoplasmic mRNAPEPCK. The inhibitory effect of insulin was readily reversible, as cells regained the basal level of mRNAPEPCK and full responsiveness to cAMP within 1 h after removing insulin. The transcript elongation assay was used to show that insulin inhibits transcription of the gene coding for mRNAPEPCK. The concentration of insulin required for 50% inhibition was 2-5 pM, whereas approximately 200 pM of proinsulin was required to achieve the same inhibition of transcription. This effect was specific, since insulin did not affect the synthesis of total RNA; it was rapid, as 5 nM insulin decreased the rate of P-enolpyruvate carboxykinase gene transcription by 50% within 15 min; and it also does not require ongoing protein synthesis. The magnitude and kinetics of the response suggest that the primary action of insulin in the regulation of P-enolpyruvate carboxykinase synthesis is exerted at the level of mRNAPEPCK transcription. The insulin-mediated inhibition of mRNAPEPCK transcription was noted in untreated cells and in cells first treated with 8-(4-chlorophenylthio)-cAMP, dexamethasone, or both of these agents. Hence, among these compounds, insulin is the dominant regulatory molecule.

Animals↗
PubMed1984

Polymorphism in the 5' flanking region of the human insulin gene. Relationships with noninsulin-dependent diabetes mellitus, glucose and insulin concentrations, and diabetes treatment in the Pima Indians.

Variations in DNA sequences flanking the insulin gene were studied in relation to noninsulin-dependent diabetes mellitus (NIDDM) in 87 unrelated Pima Indians at least 35 yr of age. DNA was isolated from nuclei of peripheral blood leukocytes and digested with restriction endonucleases. Less variation in this region was found in Pima Indians than in other racial groups previously studied. Only two classes of alleles (classes 1 and 3) were found, and there was virtually no variation within classes. At least one class 3 allele was found in 47% of the 38 nondiabetic subjects and in 37% of the 49 with NIDDM (odds ratio = 0.65, P = 0.4, 95% confidence interval for the odds ratio = 0.25 to 1.67). Homozygosity for class 3 alleles, however, was found only in diabetics. There were no differences according to genotype in obesity, fasting or postload glucose or insulin concentrations, or in the relationships between insulin and glucose concentrations. 61% (11/18) of the diabetics with a class 3 allele were receiving drug treatment for diabetes compared with only 26% (8/31) of diabetics without a class 3 allele (P = 0.03). The insulin gene polymorphism probably plays no important role in the genesis of NIDDM in Pima Indians, nor does it influence the glucose or insulin concentrations or their relationship to each other, but the class 3 allele, especially when homozygous in this population, may influence the severity of the disease as indicated by need for drug treatment.

Adult↗
PubMed1982

Insulin decreases phosphoenolpyruvate carboxykinase (GTP) mRNA activity by a receptor-mediated process.

The mRNA that codes for phosphoenolpyruvate carboxykinase accounts for approximately 0.2% of the protein synthesized in H4IIEC3 hepatoma cells maintained for 24 h in serum-free medium containing N6,O2'-dibutyryl cAMP and theophylline. This value decreases to 0.04% within 3 h after the addition of insulin. Maximal effects are produced by 10(-10) M insulin, and half-maximal deinduction of both the relative rate of synthesis of P-enolpyruvate carboxykinase and mRNA coding for P-enolpyruvate carboxykinase activity occurs at approximately 2 X 10(-12) M insulin. Porcine proinsulin is 4% as potent as porcine insulin since half-maximal deinduction of mRNA coding for P-enolpyruvate carboxykinase occurs at 5 X 10(-11) M. The concentration of proinsulin required to inhibit 125I-insulin binding by 50% is 2 X 10(-7) M, as compared to 6 X 10(-9) M for insulin; thus, the decreased sensitivity of this deinduction to proinsulin parallels the decreased binding affinity H4IIEC3 cells have for proinsulin as compared to insulin. These data indicate that insulin regulates P-enolpyruvate carboxykinase synthesis through a receptor-mediated process, that the effect occurs when less than 2% of the insulin receptors are occupied, and that this effect is exerted prior to the level of mRNA translation.

Animals↗
PubMed1980

Mitochondrial metabolism of (D,L)-threo-9, 10-dibromo plamitic acid.

Bromination of palmitoleic or palmitelaidic acid proceeds by trans addition and yields dibrominated products which cannot undergo beta-oxidation when incubated with mitochondria isolated from hamster brown adipose tissue. These mitochondria were selected because they have a high capacity for oxidation of C16 fatty acids and because they are readily uncoupled by an excess of free fatty acids of this chain length. The only metabolites which could be recovered from the incubation mixtures were dibromopalmitoylcarnitine and dibromopalmitoyl CoA. Free fatty acid was also recovered. Addition of synthetic carnitine or CoA esters of brominated fatty acids did not interfere with subsequent oxidation of palmitoylcarnitine. Addition of the free brominated fatty acids did not significantly increase the rate of oxidation of subsequent additions of palmitoylcarnitine, as did other known synthetic uncouplers. These results are consistent with observations by others that feeding brominated oils leads to brominated fatty acid incorporation into tissue lipids, and indicate why this is so. They also provide a possible explanation for the hepatic damage noted in feeding experiments.

Adipose Tissue, Brown↗
PubMed1977

Translation in Escherichia coli mini-cells containing hamster mitochondrial DNA-Co1E1 - Ampr recombinant plasmids.

Hamster mitochondrial DNA is cleaved into two fragments (4.2 and 11.4 kilobase pairs of DNA (kb)) by the restriction enzyme, Eco RI. Recombinant DNA molecules formed in vitro between an Escherichia coli plasmid, Co1E1 - Ampr, and Eco RI-digested hamster mitochondrial DNA were transformed into E. coli K12. The translation products of the parent plasmid, Co1E1 - Ampr, and recombinant plasmid DNAs containing (i) the 4.2 kb mitochondrial DNA fragment and (ii) the 11.4 kb fragment were characterized on sodium dodecyl sulfate-polyacrylamide gels using bacterial mini-cell lysates. The Co1E1 - Ampr plasmid specifies at least six polypeptides whose structural genes comprise 56% of the plasmid DNA. Insertion of hamster mitochondrial DNA at the Eco RI site of the plasmid alters the relative rate of synthesis of these six polypeptides and induces the occurrence of a new band on sodium dodecyl sulfate-polyacrylamide gels which is probably not specified by the inserted mitochondrial DNA sequences.

Ampicillin↗