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D K Granner

Publications and source records attributed to D K Granner.

At least 55 records · Page 3Linked to original sources
PubMed1996

Overexpression of hexokinase II in transgenic mice. Evidence that increased phosphorylation augments muscle glucose uptake.

Hexokinase II (HKII) is the predominant isozyme expressed in peripheral insulin-responsive tissues. To explore the role of HKII in muscle glucose metabolism, two lines of transgenic mice were generated where overexpression was restricted to striated muscle; HKII protein levels and activity were increased by 3-8-fold. Oral glucose tolerance, intravenous insulin tolerance, and insulin and lactate levels were unaffected in transgenic mice. There was a trend toward increased levels of muscle glycogen; however, glucose-6-phosphate levels were increased by 43% in transgenic skeletal muscle following in vivo glucose and insulin administration. Using 2-[3H]deoxyglucose as a tracer, in vitro basal and insulin-stimulated glucose uptake were determined in extensor digitorum longus, soleus, and epitrochlearis muscles. Maximal insulin-stimulated glucose uptake was increased by 17% (extensor digitorum longus), 34% (soleus), and 90% (epitrochlearis) in transgenic muscles; basal and submaximal glucose uptake was also modestly increased in soleus and epitrochlearis. These data suggest that increased muscle HKII (corresponding to the upper end of the physiologic range) may not be sufficient to augment net in vivo glucose homeostasis. However, glucose phosphorylation can represent a rate-limiting step for skeletal muscle glucose utilization since muscle glucose-6-phosphate levels are increased during in vivo hyperinsulinemia and hyperglycemia; furthermore, basal and insulin-mediated muscle glucose uptake can be increased by a selective increase in HKII expression.

Amino Acid Sequence↗
PubMed1996

Identification and charaterization of the second retinoic acid response element in the phosphoenolpyruvate carboxykinase gene promoter.

A previously characterized retinoic acid response element (RARE1) in the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter confers approximately 50% of the response of this gene to retinoic acid (RA). Transient transfection experiments were performed using constructs containing progressive 5' deletions of the PEPCK promoter to locate other elements that contribute to the RA response. A second RARE (RARE2) was located between -402 and -306. Methylation interference and mobility gel shift assays indicated that RAR/RXR bound specifically to a segment of DNA located between -337 and -321. This region contains consensus and degenerate half-sites for receptor binding separated by 5 bp. Mutations in either half-site selectively decreased the RA response and diminished RAR/RXR binding in mobility gel shift assays. When both RARE1 and RARE2 were mutated, 80% of the RA response was lost. Finally, RARE2 conferred a RA response in a heterologous promoter context. We conclude that RAR/RXR binds to RARE2, and that this DR5-type element is a major contributor to the response of the PEPCK gene to RA.

Animals↗
PubMed1996

New connections in the regulation of PEPCK gene expression by insulin.

Phosphoenolpyruvate carboxykinase (PEPCK) catalyses the rate-limiting step in hepatic gluconeogenesis. Glucagon (via the second messenger cAMP) and glucocorticoids stimulate transcription of the PEPCK gene whereas insulin and phorbol esters have a dominant inhibitory effect. Wortmannin, an inhibitor of 1-phosphatidylinositol 3-kinase (PI 3-kinase), blocks the inhibition of glucocorticoid- and cAMP-stimulated PEPCK gene transcription by insulin. By contrast, although phorbol esters mimic the action of insulin on the regulation of PEPCK gene transcription, wortmannin does not block the effect of these agents. Thus PI 3-kinase is required for the regulation of PEPCK gene expression by insulin but not by phorbol esters. In liver cells, insulin administration stimulates the activity of multiple protein kinases, including the p42/p44 Mitogen Activated Protein (MAP) kinase and the p70/p85 ribosomal protein S6 kinase. Selective inhibition of the activation of either kinase, utilizing the compounds PD98059 and rapamycin respectively, does not affect insulin regulation of PEPCK gene transcription. Thus regulation of PEPCK gene transcription requires PI 3-kinase but does not require the activation of either p42/p44 MAP kinase or p70/p85 ribosomal protein S6 kinase.

Gene Expression Regulation, Enzymologic↗
PubMed1996

Induction of the glucokinase gene by insulin in cultured neonatal rat hepatocytes. Relationship with DNase-I hypersensitive sites and functional analysis of a putative insulin-response element.

Previous, in vivo experiments have shown that an appropriate hormonal environment (high plasma insulin, low plasma glucagon) was unable to induce the accumulation of glucokinase mRNA in term fetal rat liver, whereas it was very efficient in the newly born rat. We have confirmed in the present study that insulin induced the accumulation of glucokinase mRNA in cultured hepatocytes from 1-day-old newborn rats, but not in cultured hepatocytes from 21-day-old fetuses. To identify regulatory regions of the glucokinase gene involved in the insulin response, we have scanned the glucokinase locus for DNase I hypersensitive sites in its in vivo conformation. We confirmed the presence of four liver-specific DNase I hypersensitive sites located in the 5' flanking region of the gene. Moreover, two additional hypersensitive sites, located at 2.5 kb and 3.5 kb upstream of the cap site were found but none of these new sites displayed inducibility by insulin. Finally, an increase of the sensitivity of hypersensitive site-1 and hypersensitive site-2 to DNase I correlates with the ability of insulin to induce glucokinase gene expression in cultured hepatocytes from 1-day-old rats, as observed in previous in vivo studies. This suggests that neither a prior exposure to insulin nor a simple aging of the fetal cells in the presence of the hormone in culture are instrumental for the full DNase-I hypersensitivity of the two proximal sites necessary for the neonatal response of the glucokinase gene to insulin. The proximal hypersensitive site-1, which is close to the transcription start site in the liver, does coincide with a sequence (designated IRSL) that is 80% identical to the phosphoenolpyruvate carboxykinase IRS and with a DNase-I footprint that has been identified overlapping this sequence. Nevertheless, functional analysis of this sequence suggested that it is unlikely that the insulin-response sequence like alone is sufficient to mediate the transcriptional effect of insulin on the hepatic glucokinase gene.

Animals↗
PubMed1996

Functional organization of mammalian hexokinase II. Retention of catalytic and regulatory functions in both the NH2- and COOH-terminal halves.

The mammalian hexokinase (HK) family includes three closely related 100-kDa isoforms (HKI-III) that are thought to have arisen from a common 50-kDa precursor by gene duplication and tandem ligation. Previous studies of HKI indicated that a glucose 6-phosphate (Glu-6-P)-regulated catalytic site resides in the COOH-terminal half of the molecule and that the NH2-terminal half contains only a Glu-6-P binding site. In contrast, we now show that proteins representing both halves of human and rat HKII have catalytic activity and that each is inhibited by Glu-6-P. The intact enzyme and the NH2- and COOH-terminal halves of the enzyme each increase glucose utilization when expressed in Xenopus oocytes. Mutations corresponding to either Asp-209 or Asp-657 in the intact enzyme completely inactivate the NH2- and COOH-terminal half enzymes, respectively. Mutation of either of these sites results in a 50% reduction of activity in the 100-kDa enzyme. Mutation of both sites results in a complete loss of activity. This suggests that each half of the HKII molecule retains catalytic activity within the 100-kDa protein. These observations indicate that HKI and HKII are functionally distinct and have evolved differently.

Animals↗
PubMed1996

Insulin regulation of phosphoenolpyruvate carboxykinase gene expression does not require activation of the Ras/mitogen-activated protein kinase signaling pathway.

Expression of phosphoenolpyruvate carboxykinase (PEPCK), the rate-limiting step in hepatic gluconeogenesis, is primarily regulated at the level of gene transcription. Insulin and phorbol esters inhibit basal PEPCK transcription and antagonize the induction of PEPCK gene expression by glucocorticoids and glucagon (or its second messenger cAMP). Insulin activates a signaling cascade involving Ras --> Raf --> p42/p44 mitogen-activated protein (MAP) kinase kinase (MEK) --> p42/p44 MAP kinase (ERK 1 and 2). Recent reports suggest that activation of this Ras/MAP kinase pathway is critical for the effects of insulin on mitogenesis and c-fos transcription but is not required for insulin action on metabolic processes such as glycogen synthesis, lipogenesis, and Glut-4-mediated glucose transport. We have used three distinct approaches to examine the role of the Ras/MAP kinase pathway in the regulation of PEPCK transcription by insulin in H4IIE-derived liver cells: (i) chemical inhibition of Ras farnesylation, (ii) infection of cells with an adenovirus vector encoding a dominant-negative mutant of Ras, and (iii) use of a chemical inhibitor of MEK. Although each of these methods blocks insulin activation of MAP kinase, none alters insulin antagonism of cAMP- and glucocorticoid-stimulated PEPCK transcription. Although phorbol esters activate MAP kinase and mimic the effects of insulin on PEPCK gene transcription, inhibition of MEK has no effect on phorbol ester inhibition of PEPCK gene transcription. Using the structurally and mechanistically distinct phosphatidylinositol 3-kinase (PI 3-kinase) inhibitors, wortmannin and LY 294002, we provide further evidence supporting a role for PI 3-kinase activation in the regulation of PEPCK gene transcription by insulin. We conclude that neither insulin nor phorbol ester regulation of PEPCK gene transcription requires activation of the Ras/MAP kinase pathway and that insulin signaling to the PEPCK promoter is dependent on PI 3-kinase activation.

Alkyl and Aryl Transferases↗
PubMed1996

Variant sequences of the Hexokinase II gene in familial NIDDM.

UNLABELLED: Hexokinase II (HKII) plays a central role in the intracellular metabolism of glucose in skeletal muscle, catalysing the phosphorylation of glucose to glucose 6-phosphate. It is therefore considered to be a potentially important candidate gene in the development of insulin resistance and non-insulin-dependent diabetes mellitus (NIDDM). The aim of this study was to screen the HKII gene for mutations in NIDDM subjects from insulin-resistant families. Insulin sensitivity was assessed in unaffected first degree relatives from families with two or more living NIDDM subjects, and 15 families were identified as being insulin resistant. In 15 NIDDM subjects (one from each family) and 4 normoglycaemic control subjects, all 18 exons of the HKII gene were amplified by the polymerase chain reaction, and the products screened for mutations using a combination of single-stranded conformational polymorphism analysis and direct sequencing. Six sequence variations were detected in the NIDDM subjects; four silent polymorphisms [GAT vs GAC at codon 251 in exon 7, AAT vs AAC at codon 692 in exon 15, CCG vs CCC at codon 736 in exon 15, and CTG vs CTA at codon 766 in exon 16]; a single base change [T-->C], 22 base pairs distal to the exon-intron junction of exon 17 in the 5'-splice donor; and a single amino acid substitution [Gln142-->His] in exon 4, which was identified in 6 of the 15 NIDDM subjects. The frequency of the mutated codon 142 allele however, was comparable between NIDDM subjects with familial NIDDM (n = 56) and normoglycaemic control subjects (n = 48) (18.8% and 14.6% for NIDDM subjects and control subjects respectively; chi 2 = 0.6, p > 0.25). In addition, measures of insulin sensitivity were comparable in normal glucose tolerant subjects with (n = 20) and without (n = 40) the codon 142 polymorphism. IN CONCLUSION: (1) mutations in the coding regions of the HKII gene are unlikely to be major determinants in the development of insulin resistance and familial NIDDM; although (2) the influence of the codon 142 mutation in combination with other abnormalities of the insulin-signalling pathway on insulin action remain to be addressed.

Amino Acid Sequence↗
PubMed1996

A novel (TA)n polymorphism in the hexokinase II gene: application to noninsulin-dependent diabetes mellitus in the Pima Indians.

Hexokinase II, one member of a family of structurally similar enzymes that catalyze the phosphorylation of glucose in the 6-position, has been suggested to play a role in the pathophysiology of noninsulin-dependent diabetes mellitus (NIDDM). The gene for hexokinase II, HK2, has been previously mapped to human chromosome 2p13 by fluorescence in situ hybridization, and two-point linkage analysis has placed it near the locus for transforming growth factor alpha, TGFA. We now report the characterization of a (TA)n polymorphism in intron 12 of HK2. Using multipoint analysis of CEPH family genotypes, we have determined the most likely locus order to be cen-D2S169-[D2S286-HK2]-[D2S145-D2S291]-[+ ++D2S45-D2S101-TGFA]-tel. As HKII is a candidate gene that could contribute to the manifestation of insulin resistance and NIDDM, we genotyped 1152 Pima Indians, a Native American tribe that has the highest reported prevalence of NIDDM in the world. Although we did not detect any linkage or association of HK2 with insulin resistance or NIDDM in the Pima Indians, the polymorphism and detailed mapping of HK2 described in this report should prove useful in the assessment of the role of this gene in the predisposition to NIDDM in other populations.

Base Sequence↗
PubMed1996

Transcription of the rat skeletal muscle hexokinase II gene is increased by acute exercise.

A single bout of acute exercise increases hexokinase (HK) II mRNA and enzyme activity [R. M. O'Doherty, D. P. Bracy, H. Osawa, D. H. Wasserman, and D. K. Granner. Am. J. Physiol. 266 (Endocrinol. Metab. 29): E171-E178, 1994]. The present study addresses the mechanism of the increase in HK II mRNA. Male rats undertook a single bout of treadmill exercise and were then killed immediately or after a predetermined recovery period. The gastrocnemius/plantaris muscle complex, composed of mixed fiber types, was excised; the nuclei were isolated; and HK I, HK II, beta-actin, and alpha-tubulin gene transcription rates were measured. Genomic DNA and plasmid DNA were used as positive and negative controls, respectively. Immediately after the cessation of 30, 45, or 90 min of exercise, HK II gene transcription rates were 1.3 +/- 0.3-,2.9 +/- 0.3-, and 4.0 +/- 0.6-fold, respectively, above those of sedentary controls. The increases after 45 and 90 min of exercise were statistically significant (P < 0.01). One hour after the cessation of 30 min of exercise, HK II gene transcription was significantly increased (1.40 +/- 0.03-fold; P < 0.05). At all time points, transcription of the HK I, beta-actin, and alpha-tubulin genes was unchanged. We conclude that the exercise-induced increase in HK II gene transcription appears to play a major role in the increase of HK II mRNA and activity.

Actins↗
PubMed1996

Regulation of gene expression by insulin.

While insulin has long been known to modulate intracellular metabolism by altering the activity or intracellular location of various enzymes, it is only in the past 10 years that the regulation of gene expression by insulin has been recognized as a major action of this hormone. This review principally focuses on the regulation of gene transcription by insulin, although recent progress in the understanding of insulin-regulated mRNA stability and translation is also summarized. The identification of cis-acting elements and associated trans-acting factors through which insulin either increases or decreases the transcription of specific genes is reviewed in detail. Recent advances in the understanding of the mechanisms of insulin signaling are discussed in the context of insulin-regulated gene transcription, and emphasis is placed on the gaps that remain between the upstream signaling molecules and the downstream trans-acting factors whose binding/transactivation potential is ultimately regulated. Finally, potential gene expression defects that may contribute to the pathophysiology of non-insulin-dependent diabetes mellitus and hypertriglyceridemia are considered.

Animals↗
PubMed1996

Examination of the phosphoenolpyruvate carboxykinase gene promoter in patients with noninsulin-dependent diabetes mellitus.

Expression of phosphoenolpyruvate carboxykinase (PEPCK), a rate-limiting enzyme in gluconeogenesis, is under dominant negative regulation by insulin. In this study, we sought to test the hypothesis that mutations in the PEPCK gene promoter may impair the ability of insulin to suppress hepatic glucose production, thereby contributing to both the insulin resistance and increased rate of gluconeogenesis characteristic of NIDDM. The proximal PEPCK promoter region in 117 patients with noninsulin-dependent diabetes mellitus and 20 obese Pima Indians was amplified by PCR and analyzed with single strand conformation polymorphism techniques. In addition, limited direct DNA sequencing was performed on the insulin response sequence and flanking regions. No DNA sequence polymorphisms were found in any patient. This result suggests that mutations in cis-acting PEPCK gene regulatory elements do not constitute a common cause of noninsulin-dependent diabetes mellitus. The significance of genetic variation in promoter regions to human disease is discussed.

Adolescent↗
PubMed1996

Hepatic nuclear factor 3 is an accessory factor required for the stimulation of phosphoenolpyruvate carboxykinase gene transcription by glucocorticoids.

Transcription of the hepatic phosphoenolpyruvate carboxykinase gene is stimulated by glucocorticoids and inhibited by insulin. The glucocorticoid response is mediated by a complex glucocorticoid response unit that consists of two glucocorticoid receptor (GR)-binding sites (GR1 and GR2) and two accessory factor-binding sites (AF1 and AF2). The complete unit is required for the full glucocorticoid response. The dominant insulin effect is mediated in part through an insulin response sequence that is coincident with the AF2 element. Members of the hepatic nuclear factor 3 (HNF3) and CCAAT enhancer binding protein (C/EBP) families bind to the AF2 element; however, there is no correlation between binding of these factors and the ability of the AF2 element to mediate an insulin response. We show here that binding of HNF3 does correlate with the stimulation of the glucocorticoid response by the AF2 element and that C/EBP is apparently not involved in this effect. This requirement for HNF3 is quite specific since the substitution of elements known to enhance the action of the GR in other promoters fails to recapitulate AF2 accessory factor activity. By contrast, an HNF3-binding site from the transthyretin gene is able to substitute for the wild type AF2 sequence and elicit a maximal glucocorticoid response. Based on current and previous observations, the glucocorticoid response unit consists of four DNA elements that bind four different proteins. These are: AF1 (hepatic nuclear factor 4/chicken ovalbumin upstream promoter transcription factor), AF2 (HNF3), GR1 (GR), and GR2 (GR).

Animals↗
PubMed1995

Structural and functional analysis of the human phosphoenolpyruvate carboxykinase gene promoter.

Phosphoenolpyruvate carboxykinase (PEPCK) catalyses the rate limiting step in hepatic and renal gluconeogenesis. Glucagon (acting via cyclic AMP (cAMP)) and glucocorticoids stimulate PEPCK gene transcription, whereas insulin has the opposite effect. Since these are the major regulatory hormones controlling glucose homeostasis, and because increased hepatic glucose production is one of the characteristics of non-insulin dependent diabetes mellitus (NIDDM), investigators have speculated that the regulation of PEPCK gene expression may be defective in patients with NIDDM. To begin to investigate this possibility we have isolated and sequenced the human PEPCK gene promoter. In addition, we have constructed and analyzed a human PEPCK promoter-chloramphenicol acetyltransferase (CAT) fusion gene in an effort to correlate differences between the rat and human promoter sequences and the hormonal regulation of transcription.

Base Sequence↗
PubMed1995

Expression cloning of a zinc-finger cyclic AMP-response-element-binding protein.

In response to specific extracellular signals, intracellular cyclic AMP levels increase, leading to a variety of responses including the alteration of transcription of many eukaryotic genes. This transcriptional effect is frequently mediated through the cyclic AMP-response element (CRE) motif T(T/G)ACGTCA. Using an expression screening approach we have cloned a yeast gene, MSN2, that encodes a 78 kDa protein that recognizes this consensus CRE motif. Phosphorylation of the MSN2 protein by the catalytic subunit of protein kinase A stimulates DNA binding in vitro. Two putative Cys2His2-type zinc fingers present in the C-terminal 79 amino acids of the MSN2 protein are sufficient to confer CRE-binding specificity. Therefore, MSN2 represents a novel CRE-binding protein distinct from the multiple previously characterized basic region-leucine zipper repeat CRE-binding proteins.

Bacteriophage lambda↗
PubMed1995

Isolation, characterization and chromosomal localization of a human pseudogene for hexokinase II.

A processed pseudogene for hexokinase II (HKII), the first such reported for a member of the hexokinase gene family, was isolated from a human genomic library by using a rat HKII cDNA as a probe. The pseudogene contains a region that is identical to the open reading frame of the human HKII cDNA at 97% of the nucleotide positions, but it contains several frameshift mutations, small deletions and insertions, and several stop codons. The human HKII pseudogene is located on the X chromosome and is integrated into a long interspersed nuclear repetitive DNA element (LINE). We estimate that this integration event occurred approximately 14-16 Myr (million years) ago.

Animals↗
PubMed1995

The role of the TATA box in the hormonal regulation of phosphoenolpyruvate carboxykinase gene expression.

In this study, we analyzed the role of the TATA box in the regulation of the phosphoenolpyruvate carboxykinase (PEPCK) gene expression by dexamethasone (DEX), retinoic acid (RA), glucagon (via cAMP) and insulin (INS). The PEPCK TATA box (TATTTAAA) was absolutely required for both basal promoter activity and hormone-mediated transactivation. However, the relative induction of PEPCK gene expression by DEX, RA and cAMP, and its repression by INS, remained unaltered despite the substitution of the PEPCK TATA box with TATA elements from the herpes simplex virus-thymidine kinase gene, gene 33 or a consensus TATA box sequence, TATAAA. The results indicate that the TATA box serves a permissive, but not defining, function in the response of the PEPCK gene to hormones, and that this function can be equally facilitated by heterologous TATA box elements.

Animals↗