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H C Towle

Publications and source records attributed to H C Towle.

At least 55 records · Page 3Linked to original sources
PubMed1991

Glucose regulates leucine-induced insulin release and the expression of the branched chain ketoacid dehydrogenase E1 alpha subunit gene in pancreatic islets.

Much evidence has accumulated to support the idea that leucine can stimulate insulin release by allosterically activating glutamate dehydrogenase thus enhancing glutamate metabolism. It is less clear how the metabolism of leucine itself contributes to the signal for insulin release. We recently found that culturing pancreatic islets for 1 day at low glucose (1 mM) suppressed glucose-induced insulin release, but preserved leucine-induced insulin release. When islets were cultured at high glucose (20 mM), glucose-induced insulin release was preserved, but leucine-induced insulin release was suppressed (MacDonald, M. J., Fahien, L. A., McKenzie, D. I., and Moran, S. M. (1990) Am. J. Physiol., 259, E548-E554). The suppression of leucine-induced insulin release can be explained by glucose's suppression of the synthesis of the enzyme that catalyzes the first committed step of leucine metabolism, branched chain ketoacid dehydrogenase complex (BCKDH). High glucose suppressed the enzyme activity of the E1 component of the BCKDH complex, as well as the total activity of the BCKDH complex, to usually negligible levels in islets and decreased by an average of 90% the mRNA which encodes E1 alpha, the catalytic subunit of the E1 component of BCKDH, in islets and rat insulinoma cells. Time course studies showed that about 24 h in culture was required to maximally induce or suppress the expression of BCKDH E1 alpha. Culture at high glutamine with or without leucine mimicked to a lesser and more variable degree the effects of high glucose on leucine-induced insulin release and BCKDH E1 alpha mRNA. Leucine-plus-glutamine-induced insulin release was present after culture of islets with glucose and with or without any other secretagogue. Also, glutamate dehydrogenase transcripts and enzyme activity were not significantly altered by varying the concentration of glucose in the culture medium. Thus, leucine's insulinotropism via activation of glutamate dehydrogenase is constitutive. Preproinsulin mRNA levels were markedly increased at high glucose and glyceraldehyde phosphate dehydrogenase transcripts were either unaffected or slightly increased by glucose. Glutamine did not significantly effect the expression of genes other than BCKDH E1 alpha, and leucine had little or no effect on the expression of any of the four genes.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

An inducible nuclear factor binds to a growth hormone-regulated gene.

Transcription of the serine protease inhibitor (Spi) 2.1 gene, a member of the serine protease inhibitor family, is induced by growth hormone (GH) in rat liver. To further study the mechanism involved in this process, we have isolated and characterized the Spi 2.1 gene from a rat genomic library. Examination of the 5'-flanking region of the Spi 2.1 gene from normal animals revealed the presence of a DNase I hypersensitive site within 500 base pairs of the transcriptional initiation site, which was not detectable in hypophysectomized animals. Portions of the 5'-flanking region of the Spi 2.1 gene were fused to a heterologous promoter and reporter gene and introduced into primary rat hepatocytes by lipofection. Spi 2.1 sequences from -275 to -54 gave a 2-3-fold induction of reporter gene activity in cells grown in the presence of GH, similar to the level of induction of the endogenous Spi 2.1 mRNA in isolated hepatocytes. Further definition of the essential sequences revealed that a segment from -147 to -102 could confer GH responsiveness when linked in tandem copies in front of a heterologous promoter. Using the gel shift assay, a nuclear factor(s) from normal rat liver was identified which could interact with this minimal response fragment. The importance of this activity to GH regulation was suggested by the fact that it was absent in hypophysectomized animals but reappeared by 1 h after treatment of such animals with GH. The appearance of this activity was not blocked by pretreatment of animals with an inhibitor of protein synthesis, suggesting a preexisting factor is modified by GH to yield an activity which interacts with the Spi 2.1 gene.

Animals↗
PubMed1990

Identification of multiple thyroid hormone response elements located far upstream from the rat S14 promoter.

Expression of the rat gene designated S14 is rapidly and markedly induced by 3,3',5-triiodo-L-thyronine (T3) in the liver. We have used nuclear run-on assays and transfection studies to explore the basis of this regulation. These studies indicate that T3 induction of hepatic S14 mRNA is due primarily, if not exclusively, to changes in the rate of gene transcription. Both alpha and beta forms of the thyroid hormone receptor (c-erbA) can activate S14 promoter activity in a co-transfection assay. This activation occurs in a dose-dependent manner and requires the expression of the T3 receptor from transfected cDNA in either COS-1 cells or primary rat hepatocytes. Delineation of sequences essential for control of S14 promoter activity indicates that multiple thyroid hormone response elements (TREs) are involved. These TREs have been localized in the far upstream 5'-flanking region of the S14 gene, approximately 2.7 kilobases from the S14 transcriptional initiation site. DNA-binding studies with in vitro translated c-erbA indicate that each of the S14 TREs contains a specific binding site for the receptor. The location of the S14 TREs far upstream from the promoter site may necessitate the presence of multiple TREs for hormonal responsiveness.

Animals↗
PubMed1989

Sequences within the 5'-flanking region of the S14 gene confer responsiveness to glucose in primary hepatocytes.

The DNA sequences involved in control of S14 gene expression in response to carbohydrate have been studied. The levels of S14 mRNA in primary hepatocytes increase when glucose in the media is elevated from 5.5 to 27.7 mM in the presence of insulin. Following lipofection of primary hepatocytes, plasmids containing S14 genomic sequences from -4316 to +19 relative to the start of transcription were sufficient to confer glucose regulation to the linked marker gene, chloramphenicol acetyltransferase. Deletions of the S14 sequences between -4316 and -1601 led to a significant reduction in glucose-stimulated activity with each successive deletion, suggesting the presence of multiple regulatory elements. The response of the transfected construct containing 4316 base pairs of S14 5'-flanking region mimicked changes in the endogenous S14 mRNA levels in all hormonal and nutritional conditions tested, supporting the physiological significance of the response.

Animals↗
PubMed1989

Identification of nuclear factors that enhance binding of the thyroid hormone receptor to a thyroid hormone response element.

Using a gel shift assay, we analyzed the binding of in vitro translated alpha- and beta-thyroid hormone (T3) receptors to a T3-response element (TRE) derived from the rat GH gene. No receptor-TRE complexes were observed when translated receptor alone was incubated with the TRE. However, addition of a nuclear extract from liver to the translational products resulted in the formation of two receptor-DNA complexes for both the alpha- and beta-receptors. These complexes were shown to contain translated receptor by comigration of 32P-labeled TRE and 35S-labeled receptor in the gel shift assay. A competition experiment demonstrated that formation of the complexes was sequence specific. Preincubation of the liver nuclear extract at 60 C abolished formation of both complexes indicating that receptor-TRE binding required a heat-labile nuclear factor. Phosphocellulose chromatography of the nuclear extract resulted in separation of the activities required for formation of the two complexes. Analysis of nuclear extracts from different tissues revealed that one complex formed in the presence of all extracts, whereas the second complex appeared predominantly with a nuclear extract from liver. Addition of T3 to the binding reaction had no effect on receptor-TRE complex formation. We suggest that nuclear factors interact with the T3 receptor to enhance hormone-independent binding to a TRE.

Binding, Competitive↗
PubMed1988

Isolation and characterization of rat cDNA clones for two distinct thyroid hormone receptors.

Two distinct v-erbA-related cDNA clones representing the products of different genes were isolated from a rat liver cDNA library. The first, rc-erbA-alpha, was 82% identical to v-erbA and encoded a polypeptide with a calculated molecular mass of 45,000 daltons. This cDNA clone arises from the same gene product as a v-erbA-related cDNA isolated from rat brain by Thompson et al. (Thompson, C. C., Weinberger, C., Lebo, R., and Evans, R. (1987) Science 237, 1610-1614). The second cDNA clone, rc-erbA-beta, was 76% identical to v-erbA and encoded a polypeptide with a calculated molecular mass of 52,000 daltons. Both rc-erbA-alpha and rc-erbA-beta translational products bound 3,5,3'-triiodo-L-thyronine with affinities equal to each other (Kd approximately equal to 0.4 nM) and comparable to the nuclear thyroid hormone receptor extracted from rat liver. The relative affinities of a series of thyroid hormone analogs for both translational products were also identical. In various tissues and cell lines, the relative levels of rc-erbA-beta RNA, but not rc-erbA-alpha RNA, correlated with measurements of nuclear 3,5,3'-triiodo-L-thyronine binding sites. Based on this correlation, we suggest that rc-erbA-beta may encode the "classical" nuclear thyroid hormone receptor, whereas rc-erbA-alpha may encode an isoreceptor species with differing functional properties.

Amino Acid Sequence↗
PubMed1988

Direct effects of endotoxin on hepatocytes. Synthesis of a specific secretory protein.

The synthesis of acute-phase proteins by the liver during sepsis has been thought to be induced primarily by monokines released from activated macrophages, although glucocorticoid hormones may also stimulate this process to a lesser degree. According to this concept, synthesis of these proteins following administration of bacterial endotoxins would be an indirect effect and would not reflect a direct interaction of the endotoxin molecule with the hepatic parenchymal cell. We observed, however, that the synthesis of a 23-kilodalton protein was stimulated directly by the addition of lipopolysaccharide to cultures of primary mouse hepatocytes. The synthesis of this protein was also stimulated by glucocorticoids and interleukin 1. These findings demonstrate that certain hepatic proteins are subject to complex regulation by several factors thought to be important mediators of sepsis; in addition, they suggest that hepatic parenchymal cells may have the intrinsic capacity to respond directly to bacterial endotoxins.

Animals↗
PubMed1987

Growth hormone induces two mRNA species of the serine protease inhibitor gene family in rat liver.

In order to study the molecular actions of growth hormone on gene expression, we have cloned and characterized two unique, but related, cDNA sequences from rat liver, lambda Spi-1 and lambda Spi-2. These two cDNA sequences are complementary to rat hepatic mRNA species previously designated as Spots 3 and 20 when assayed by in vitro translation and two-dimensional gel electrophoresis. By Northern blot, the two mRNAs are both 1900 bases in length and growth hormone administered to hypophysectomized rats increases the levels of both of these mRNAs. In contrast, the combined administration of thyroxine, corticosterone, and dihydrotestosterone to hypophysectomized rats did not augment these mRNAs. The simultaneous administration of all four hormones resulted in a level greater than that observed for animals treated with growth hormone alone. Analysis of genomic DNA suggests the presence of two similar, but not identical, genes. DNA sequencing of lambda Spi-1 and lambda Spi-2 revealed that they were 90% homologous at the nucleotide level and 87% homologous at the amino acid sequence level. lambda Spi-2 has 78% homology with mouse contrapsin, 60% with human alpha 1-antichymotrypsin, and 51-55% with alpha 1-antitrypsins, all members of the serine protease inhibitor gene family. The nucleotide and deduced amino acid sequences of lambda Spi-1 and lambda Spi-2 which align with the reactive centers of known members of this family differ substantially from each other and from other members of the family. The difference in the reactive center suggests that the specificity or function of these proteins may differ from other members of serine protease inhibitor gene family.

Amino Acid Sequence↗
PubMed1987

Induction of a rapidly responsive hepatic gene product by thyroid hormone requires ongoing protein synthesis.

The regulation of a gene, designated spot 14, which is rapidly induced in rat liver in response to 3,5,3'-triiodo-L-thyronine (T3) was studied as a model for exploring the molecular basis of thyroid hormone action. The time course of induction of the nuclear precursor to spot 14 mRNA after intramuscular injection of T3 displayed a very short lag period of between 10 and 20 min. The rapidity of this effect suggests that the induction in gene expression occurs as a primary response to the hormone-receptor interaction. The protein synthesis inhibitor cycloheximide injected 15 min before T3 completely blocked the accumulation of nuclear precursor RNA 30 min after T3 treatment. Emetine, an inhibitor of protein synthesis which acts by a different mechanism than cycloheximide, also blocked the induction of the spot 14 nuclear precursor RNA. The increased rate of spot 14 gene transcription observed after T3 treatment, as measured by nuclear run-on assay, was similarly completely abolished in the presence of cycloheximide. In addition, ongoing protein synthesis was required for maintaining spot 14 nuclear precursor RNA at induced levels in animals previously treated with T3. On the other hand, cycloheximide had no effect on T3 uptake or binding to the nuclear receptor during the 45-min time frame studied. The paradox of the rapid kinetics of induction and the requirement of ongoing protein synthesis may be explained by a protein with an extremely short half-life which is necessary for T3 induction of the spot 14 gene.

Animals↗
PubMed1986

Opposing effects of glucagon and triiodothyronine on the hepatic levels of messenger ribonucleic acid S14 and the dependence of such effects on circadian factors.

We have studied the effect of glucagon on the expression of a triiodothyronine (T3) and carbohydrate-inducible mRNA sequence (mRNA-S14) in rat liver that undergoes a threefold diurnal variation (peak, 2200 h; nadir, 0800 h). Glucagon injection into euthyroid rats (25 micrograms/100 g body wt i.p., three doses at 15-min intervals) during the nocturnal plateau of mRNA-S14 caused a monoexponential disappearance of this sequence (t1/2, 90 min) accompanied by a 90% reduction in the transcriptional rate in a nuclear run-off assay, indicative of a near total reduction of synthesis. This effect was markedly attenuated in rats treated with T3 (200 micrograms/100 g body wt i.p.) 24 h before glucagon injection. When T3 was given 15 min after glucagon, the glucagon-initiated decline in mRNA-S14 was reversed within 90 min, suggesting a rapid interaction between the two hormones in the evening. Curiously, administration of T3 alone at this hour did not affect a significant increase in mRNA-S14. At 0800 h, however, T3 caused the expected brisk induction of this sequence, whereas glucagon was without effect. In essence, glucagon affected mRNA-S14 synthesis only in the evening, while T3 increased levels of this sequence above the baseline only in the morning. T3, however, reversed the effect of prior glucagon injection at night. The observed alterations in hormonal responsivity could underly the diurnal variation of mRNA-S14 expression. Moreover, the data suggest the hypothesis that T3 may act on S14 gene expression by antagonizing factors that inhibit its transcription.

Animals↗
PubMed1986

Dissociation of hepatic messenger ribonucleic acidS14 levels and nuclear transcriptional rates in suckling rats.

The messenger RNA (mRNA) coding for the rat hepatic protein spot 14(S14) (mol wt 17,000; pI 4.9) is rapidly responsive to T3 and carbohydrate administration in the adult animal. In 15-day-old suckling rats, the level of expression is 200-fold less than in 60-day-old chow-fed animals. However, the relative transcriptional activity of the S14 gene is only 30% higher in the 60-day-old rat than in the 15-day-old animal. Although T3 induces a 100-fold increase in mRNAS14 in 15-day-old animals, there was only a 17% increase in the S14 gene transcriptional activity. These results indicate that the hormonal and developmental regulation of the S14 gene in liver appears to be predominantly directed at the posttranscriptional level.

Animals↗
PubMed1986

Regulation of hepatic gene expression by lipogenic diet and thyroid hormone.

Adaptive hyperlipogenesis in the liver occurs after feeding rats a high-carbohydrate, fat-free diet or after treatment with thyroid hormone. This phenomenon is accompanied by the induction of a family of enzymes and proteins involved in various aspects of lipogenesis, resulting largely from alterations in the rates of protein synthesis. The changes in protein production, in turn, are the result of increased or decreased cellular concentrations of specific mRNAs in response to carbohydrate feeding or hyperthyroidism. There is a large degree of overlap between the mRNA species induced by thyroid hormone and carbohydrate feeding in rat liver. One such mRNA species, spot 14, has several attractive features as a potential model for exploring the molecular mechanisms involved in the regulation of gene expression. Most importantly, the mRNA for spot 14 increases with a lag time of less than 20 min after treatment with thyroid hormone or sucrose gavage and thus may represent a primary response to the hormonal and dietary stimuli. Initial studies to elucidate the cellular site of action of thyroid hormone and dietary carbohydrate indicate that changes in both the rate of gene transcription and the stability of the nuclear RNA precursor are involved in spot 14 mRNA induction.

Animals↗
PubMed1985

Translational control of the circadian rhythm of liver sterol carrier protein. Analysis of mRNA sequences with a specific cDNA probe.

The striking changes in amount of rat liver SCP (sterol carrier protein) during a 24-h dark-light cycle are due to alterations in the relative synthetic rate of SCP. However, functional SCP mRNA, measured by a cell-free translational assay, does not fluctuate in the dark-light cycle. Since cell-free translational assays do not always reflect the actual abundance of an mRNA molecule, a specific cDNA hybridization assay was used to directly quantitate SCP mRNA sequences. The cDNA probe was selected from a rat liver library by hybridization to a mixture of synthetic oligonucleotides containing a portion of the sequence of SCP mRNA. The relative amount and size distribution of the SCP mRNA species (approximately 700-800 nucleotides) does not change during the diurnal cycle. To explore possible mechanisms of this translational control, the polysomal distribution of SCP mRNA was compared at the maximum and minimum points of SCP synthesis. No significant amounts of SCP mRNA were present in nonpolysomal ribonucleoprotein particles. Furthermore, no alteration in the relative level of SCP mRNA associated with polysomes or in polysome size occurs at the maximum and minimum points of SCP synthesis. Thus, changes in total SCP mRNA levels or its polysomal distribution cannot account for the diurnal variation in SCP synthesis.

Animals↗
PubMed1985

Role of sterol carrier protein in cholesterol metabolism.

This report summarizes our recent studies on the protein known as sterol carrier protein (SCP) or fatty acid binding protein (FABP). SCP is a highly abundant, ubiquitous protein with multifunctional roles in the regulation of lipid metabolism and transport. SCP in vitro activates membrane-bound enzymes catalyzing cholesterol synthesis and metabolism, as well as those catalyzing long chain fatty acid metabolism. SCP also binds cholesterol and fatty acids with high affinity and rapidly penetrates cholesterol containing model membranes. Studies in vivo showed SCP undergoes a remarkable diurnal cycle in level and synthesis, induced by hormones and regulated in liver by translational events. SCP rapidly responds in vivo to physiological events and manipulations affecting lipid metabolism by changes in level. Thus SCP appears to be an important regulator of lipid metabolism. Preliminary evidence is presented that SCP is secreted by liver and intestine into blood and then taken up by tissues requiring SCP but incapable of adequate SCP synthesis.

Animals↗
PubMed1985

Stabilization of a specific nuclear mRNA precursor by thyroid hormone.

The regulation of a thyroid hormone-responsive gene in rats, designated spot 14, was explored. The expression of this gene in liver is rapidly (less than 10 min) and markedly (greater than 10-fold) altered by the administration of 3,5,3'-triiodo-L-thyronine (T3) to hypothyroid rats (P. Narayan, C. W. Liaw, and H. C. Towle, Proc. Natl. Acad. Sci. USA 81:4687-4691, 1984). To investigate the cellular site at which T3 acts to induce this hepatic mRNA, we made parallel measurements of the relative levels of spot 14 mRNA and nuclear precursor RNA and of the rate of gene transcription after treatments designed to alter the thyroid status of rats. The relative levels of both the mRNA and nuclear precursor were elevated roughly 5- to 6-fold in euthyroid animals and 9- to 12-fold in hyperthyroid animals over those in hypothyroid controls. However, only a small difference of approximately 1.5-fold was detected in the rate of spot 14 gene transcription. After a single injection of T3 into hypothyroid animals, a small and transient rise in the transcription rate was detected at 30 min. However, the levels of spot 14 mRNA and nuclear precursor RNA increased much more dramatically throughout the first 4 h of treatment. In both cases, changes in the rate of gene transcription were not capable of accounting for the alterations observed in mRNA levels. Thus, the major site of spot 14 gene regulation by T3 is at a posttranscriptional level. The proportional changes observed in the nuclear precursor and mRNA levels suggest that the site of control is at the level of stability of the nuclear precursor RNA for spot 14 mRNA.

Animals↗
PubMed1984

Repression of rat kidney L-arginine:glycine amidinotransferase synthesis by creatine at a pretranslational level.

The first committed reaction in the biosynthesis of creatine is catalyzed by the enzyme L-arginine:glycine amidinotransferase, commonly called transamidinase. Creatine, the end product of the biosynthetic pathway, is known to alter the levels of kidney transamidinase activity. Rats fed a diet containing 0.3% creatine had 26% of the kidney transamidinase activity of the rats fed a creatine-free diet. This reduction in transamidinase activity was correlated with a decrease in transamidinase protein in the creatine-fed rats. The relative synthetic rates and mRNA functional activities of transmidinase were measured in control and creatine-fed rats. The relative synthetic rate of transamidinase in creatine-fed rats was 21% of that found in the control animals. The functional transamidinase mRNA in creatine-fed rats was correspondingly reduced to 37% of the amount in the control animals. Thus, creatine affects transamidinase activity by altering its rate of synthesis at a pretranslational step and represents an example of end-product repression in a higher eukaryote.

Amidinotransferases↗
PubMed1984

Characterization of a thyroid hormone-responsive gene from rat.

We have isolated and characterized a rat gene coding for spot 14 mRNA: a hepatic product induced rapidly by thyroid hormone. This gene is present in a single copy/haploid genome, but encodes two mRNA species differing by 170 nucleotides in length. Through S1 nuclease analyses, the difference was mapped to the 3'-end of the mRNA with one species extending 170 nucleotides beyond the 3'-end of the other mRNA. The putative poly(A) addition signals AUUAAA and AAUAAA are found to precede the sites of polyadenylation in the shorter and longer mRNA, respectively. The mRNA codes for a protein of 150 amino acids with a molecular weight of 17,010. The protein-coding region is located closer to the 5'-end of the mRNA, leaving a relatively long 3'-untranslated region, which contains the only intron of 3,150 base pairs within the gene. At approximately 27 base pairs upstream from the start site of the mRNA, a sequence homologous to the TATA box, TAGAAAT , was found.

Amino Acid Sequence↗