Glucose and other insulin secretagogues induce, rather than inhibit, expression of Id-1 and Id-3 in pancreatic islet beta cells.

AIMS/HYPOTHESIS: Basic helix loop helix transcription factors regulate insulin gene transcription. Therefore, molecules that regulate their function should affect insulin production and secretion. As Id proteins inhibit basic helix loop helix function, it is important to determine whether they are expressed in beta cells and if insulin secretagogues regulate their expression. METHODS: Human islets or insulinoma cells were cultured in different glucose concentrations or treated with secretagogues. Insulin secretion was measured using RIA. The Id mRNA and protein concentrations were measured using northern blots, RT-PCR, and western blots. Transfections of promoter-reporter constructs were used to estimate Id-1 gene transcription. RESULTS: The Id-1 mRNA concentrations were twofold higher in islets cultured overnight in 10 mmol/l than in 2.5 mmol/l glucose. Addition of high glucose to islets previously cultured in low glucose, increased Id-1 mRNA concentrations within 30 min. Analyses using insulinoma cells revealed that Id-1 and Id-3 mRNA concentrations peaked 30 min after glucose was added, returned to near basal concentrations by 2 h and then progressively increased for 24 h. The Id-1 protein concentrations changed in a similar pattern. Insulin secretagogues that act through different signaling pathways also induced Id expression. The Id response required glucose metabolism, calcium, and RNA synthesis but not protein synthesis. Glucose-responsive elements are confined to the 5'-region of the Id-1 gene. CONCLUSION/INTERPRETATION: The concomitant induction of Id-1 and Id-3 expression, insulin gene transcription, and insulin secretion suggests that physiological concentrations of Ids do not inhibit insulin gene transcription and Ids could play unexpected and novel roles in promoting beta-cell function.

Forced expression of Id-1 in the adult mouse small intestinal epithelium is associated with development of adenomas.

Ids are dominant-negative helix-loop-helix (HLH) proteins that play overlapping yet distinct roles in antagonizing basic HLH transcription factors. Although Ids affect myogenesis, neurogenesis, and B-cell development, little is known about their in vivo functions in epithelia. We have examined the effects of forced expression of Id-1 in the small intestinal epithelium of adult chimeric mice. 129/Sv embryonic stem cells, transfected with DNA containing Id-1 under the control of transcriptional regulatory elements that function in all intestinal epithelial cell lineages, were introduced into C57Bl/6 (B6) blastocysts heterozygous for the ROSA26 marker. The B6 ROSA26/+ intestinal epithelium of the resulting adult chimeras produces Escherichia coli beta-galactosidase, allowing identification of this internal control cell population. Chimeras produced from nontransfected embryonic stem cells served as additional controls. Immunohistochemical studies of the control chimeras indicated that the small intestinal epithelium supports a complex pattern of endogenous Id expression. Id-1 is restricted to the cytoplasm; levels do not decrease as descendants of multipotent intestinal stem cells differentiate. Id-2 and Id-3 are only detectable in nuclei; levels increase markedly as epithelial cells differentiate. Forced expression of Id-1 in the 129/Sv epithelium results in a decline in Id-2 and Id-3 to below the limits of immunodetection. A subset of chimeric-transgenic mice lacked growth factor- and defensin-producing Paneth cells in their 129/Sv epithelium and also developed intestinal adenomas. These changes were not present in normal control chimeras. Adenomas were composed of proliferating beta-Gal-positive and -negative epithelial cells, suggesting that they arose through cooperative interactions between 129/Sv(Id-1) and B6 ROSA26/+ cells. These chimeras provide a model for studying how perturbations in Id expression affect tumorigenesis.

A tetraspan membrane glycoprotein produced in the human intestinal epithelium and liver that can regulate cell density-dependent proliferation.

The human cell line HT-29 provides a model system for studying regulation of proliferation and differentiation in intestinal epithelial cell lineages: (i) HT-29 cells cultured in glucose resemble undifferentiated multipotent transit cells located in the lower half of intestinal crypts; (ii) proliferating HT-29 cells cultured in inosine resemble committed cells located in the upper half of the crypt; (iii) nonproliferating, confluent HT-29-inosine cells have features of differentiated enterocytes and goblet cells that overlie small intestinal villi. A cDNA library prepared from HT-29-inosine cells was screened with a series of subtracted cDNA probes to identify proteins that regulate proliferation/differentiation along the crypt-villus axis. A cDNA was recovered that encodes a 202-amino acid protein with four predicted membrane spanning domains and two potential sites for N-linked glycosylation. Levels of this new member of the superfamily of tetraspan membrane proteins (TMPs) increase dramatically as nondividing epithelial cells exit the proliferative compartment of the crypt-villus unit and migrate onto the villus. The protein is also produced in nondividing hepatocytes that have the greatest proliferative potential within liver acini. Three sets of observations indicate that in the appropriate cellular context, intestinal and liver (il)-TMP can mediate density-associated inhibition of proliferation. (i) Accumulation of il-TMP glycoforms precedes terminal differentiation of HT-29-inosine cells and occurs as they undergo density-dependent cessation of growth. il-TMP levels are lower and glycosylation less extensive in HT-29-glucose cells, which do not undergo growth arrest at confluence. (ii) HeLa cells normally do not produce il-TMP. Forced expression of il-TMP inhibits proliferation as cells approach confluence. The extent of il-TMP glycosylation in the transfected cells is similar to that observed in HT-29-inosine cells and greater than in HT-29-glucose cells. (iii) SW480 cells are derived from a human colon adenocarcinoma and do not express il-TMP. Like nontransfected HeLa cells, they do not stop dividing at confluence, whether grown in medium containing glucose or inosine. Expression of il-TMP has no effect on the growth properties of SW480 cells. The extent of il-TMP glycosylation in SW480-glucose cells is similar to that noted in HT-29-glucose cells, lending further support to the notion that il-TMP's activity is related to its state of N-glycosylation.

A strategy for isolation of cDNAs encoding proteins affecting human intestinal epithelial cell growth and differentiation: characterization of a novel gut-specific N-myristoylated annexin.

The human intestinal epithelium is rapidly and perpetually renewed as the descendants of multipotent stem cells located in crypts undergo proliferation, differentiation, and eventual exfoliation during a very well organized migration along the crypt to villus axis. The mechanisms that establish and maintain this balance between proliferation and differentiation are largely unknown. We have utilized HT-29 cells, derived from a human colon adenocarcinoma, as a model system for identifying gene products that may regulate these processes. Proliferating HT-29 cells cultured in the absence of glucose (e.g., using inosine as the carbon source) have some of the characteristics of undifferentiated but committed crypt epithelial cells while postconfluent cells cultured in the absence of glucose resemble terminally differentiated enterocytes or goblet cells. A cDNA library, constructed from exponentially growing HT-29 cells maintained in inosine-containing media, was sequentially screened with a series of probes depleted of sequences encoding housekeeping functions and enriched for intestine-specific sequences that are expressed in proliferating committed, but not differentiated, epithelial cells. Of 100,000 recombinant phage surveyed, one was found whose cDNA was derived from an apparently gut-specific mRNA. It encodes a 316 residue, 35,463-D protein that is a new member of the annexin/lipocortin family. Other family members have been implicated in regulation of cellular growth and in signal transduction pathways. RNA blot and in situ hybridization studies indicate that the gene encoding this new annexin exhibits region-specific expression along both axes of the human gut: (a) highest levels of mRNA are present in the jejunum with marked and progressive reductions occurring distally; (b) its mRNA appears in crypt-associated epithelial cells and increases in concentration as they exit the crypt. Villus-associated epithelial cells continue to transcribe this gene during their differentiation/translocation up the villus. Immunocytochemical studies reveal that the intestine-specific annexin (ISA) is associated with the plasma membrane of undifferentiated, proliferating crypt epithelial cells as well as differentiated villus enterocytes. In polarized enterocytes, the highest concentrations of ISA are found at the apical compared to basolateral membrane. In vitro studies using an octapeptide derived from residues 2-9 of the primary translation product of ISA mRNA and purified myristoyl-CoA:protein N-myristoyltransferase suggested that it is N-myristoylated. In vivo labeling studies confirmed that myristate is covalently attached to ISA via a hydroxylamine resistant amide linkage. The restricted cellular expression and acylation of ISA distinguish it from other known annexins.(ABSTRACT TRUNCATED AT 400 WORDS)

Serum factors that stimulate fatty acid oxidation: physiological specificity.

In the accompanying paper (Wice et al., 1986) we reported that serum from chickens contains small molecular weight compounds that stimulate long-chain fatty acid oxidation ten fold or more in HeLa cells. Here we show that this response is not limited to specific sera or to specific target cells. The specificity of the metabolic response to these factors was also investigated. They had no effect on the following major pathways of HeLa cell metabolism: 1) the oxidation of the medium-chain fatty acid, octanoic acid, 2) the rate of glycolysis of glucose, 3) the flux of glucose carbon through the oxidative arm of the pentose cycle, 4) the entry of pyruvate into the citrate cycle, 5) the oxidation of glutamine carbon, 6) the utilization rate of oxygen or 7) the rate of fatty acid synthesis. Furthermore, the increased oxidation of long-chain fatty acids was not a result of an increased uptake into the cells. Thus, the serum factors appear to be very specific for the oxidation of long-chain fatty acids for energy. Since carnitine also stimulates long-chain fatty acid oxidation in these cells, it seems likely that these compounds either facilitate the activity of carnitine or provide the same function--presumably the transport of long-chain fatty acid into and out of the mitochondria.

Serum factors that stimulate fatty acid oxidation: properties of factors.

Cultured heart muscle cells, but not HeLa cells, oxidize long-chain fatty acids in medium containing dialyzed serum. Addition of chicken serum dialysate (or non-dialized serum) stimulated palmitic acid oxidation by HeLa cells 10 to 20 fold. This serum activity was not eliminated by lipid extraction, ethanol or acid precipitation, alkaline phosphatase treatment, or autoclaving. About 80% was lost after any one of the following treatments: 6N HCl at 110 degrees C for 16 hr, pepsin, Dowex cation exchange at pH 3, or 1N KOH at 100 degrees C for 1 hr. Serum activity was separated into five or more peaks by gel filtration with Sephadex G-10. Each of these peak fractions was further purified by HPLC using a cyanopropyl-bonded resin. Carnitine, which is important for the transport of long-chain fatty acids into mitochondria for oxidation, also stimulated the oxidation of palmitate. However, these serum factors are not known precursors to carnitine since its immediate precursor 4-n-trimethylaminobutyrate, did not stimulate palmitate oxidation. Total carnitine, including that in acylcarnitine compounds, was approximately 15 microM in the chicken sera to give approximately 0.7 microM in the medium. Based on the fraction of total activity accountable by carnitine and fractional stability to acid, alkali, and pepsin, about 75% of the activity is from non-carnitine compounds. Only one of the factors appears to be carnitine or an acylcarnitine derivative. Several lines of evidence suggest that the other factors are peptide compounds.

Isolating and sequencing the predominant 5'-ends of a specific mRNA in cells. I. Purification by filter hybridization.

Procedures are considered for purification of a specific procaryotic RNA by successive hybridizations to DNA immobilized to nitrocellulose with special consideration of problems associated with subsequent end-labeling in the T4 polynucleotide kinase reaction. (1) Inhibitors of the kinase can be associated with the plasmid but were removed by electrophoresis of the DNA fragment through polyacrylamide. (2) Residual soluble acrylamide, contaminating the DNA and preventing its efficient retention to nitrocellulose, could be removed by DE52 chromatography. (3) Short denatured DNA required high salt (0.9 M) to bind to nitrocellulose but reannealed quickly at those salt concentrations unless applied at less than or equal to 0.3 micrograms/ml at 4 degrees C with a flow rate of 1 ml/min. (4) The kinetics of the hybrid reaction were a function of DNA length, concentration, and temperature. (5) Formamide was a more effective denaturing agent to remove hybrid RNA from the filter than either 12 M urea or 8 M guanidine-HCl, but caused significant release of DNA from the nitrocellulose as well as another potent inhibitor of the kinase reaction. The release of DNA and other kinase inhibitors was greatly reduced by eluting in boiling water.

Isolating and sequencing the predominant 5'-ends of a specific mRNA in cells. II. End-labeling and sequencing.

T4 polynucleotide kinase has been used to end-label specific RNA purified by multiple hybridizations to nitrocellulose-bound DNA. The pico moles of ends of a specific mRNA transcribed from the chromosome, even from several liters of Escherichia coli, give concentrations perhaps 2000-fold below the Km value of the kinase-RNA substrate. In such a reaction, optimal incorporation was observed with increasing ATP concentration to greater than or equal to 7 microM (greater than or equal to 15 mCi of carrier-free [32P]ATP in a 300-500 microliter reaction). The unreacted ATP (greater than 150-fold excess) could best be eliminated by multiple gel filtrations rather than by precipitation, ion exchange chromatography or dialysis. The [5'-32P]RNA was digested with T1 or pancreatic RNase and the [5'-32P]oligonucleotides separated by size in a 20% polyacrylamide gel. Oligonucleotides of a specific size were separated sufficiently by a second dimension electrophoresis on cellulose acetate. We have used partial alkali digestion in sequencing the purified oligonucleotides. As opposed to other digestions, alkali produces 5',3'-diphospho-oligonucleotides whose mobilities can differ from those of the monophosphates, e.g., much longer running times in conventional homochromatography.

The intracellular accumulation of UDP-N-acetylhexosamines is concomitant with the inability of human colon cancer cells to differentiate.

The relationship between the intracellular concentration of various nucleotides as measured by high-performance liquid chromatography analysis, and the differentiation of 2 human colon cancer cell lines was studied. HT-29 cells were induced to undergo both structural and functional enterocytic differentiation (as determined by electron microscopy and the presence of brush-border specific enzymes, respectively) by changing the carbon source or adding Na butyrate to standard tissue culture media. This differentiation occurred after the cells reached confluency when they were cultured in galactose, uridine, inosine, or without nucleosides (all in the absence of glucose) and in the presence of glucose plus Na butyrate. Cells cultured in 25 mM fructose or glucose +/- nucleosides did not differentiate. In all culture conditions where HT-29 cells did not differentite, the intracellular concentrations of 2 compounds which co-migrated with UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine rose approximately equal to 10-fold at confluency and remained elevated throughout the stationary phase, whereas their concentrations remained constant and low after confluency in cells that underwent differentiation. This indicated that the accumulation of these compounds is associated with the inability of these cells to differentiate since other nucleotides and nucleotide sugars did not change in a similar fashion. Purification of the presumed UDP-N-acetylhexosamines, followed by the identification of the products from their chemical and enzymatic hydrolysis, confirmed the identity of these two peaks. Nucleotide analysis of Caco-2 cells, which undergo enterocytic differentiation after they reach confluency even when cultured on glucose, revealed the same pattern of UDP-N-acetylhexosamine levels as differentiated HT-29 cells, with its concentration remaining relatively constant and very low, even after the cells were confluent. The significance of the accumulation of UDP-N-acetylhexosamines in cells unable to differentiate is discussed.

Sugar-free growth of mammalian cells on some ribonucleosides but not on others.

It was shown earlier that a variety of vertebrate cells could grow indefinitely in sugar-free medium supplemented with either uridine or cytidine at greater than or equal to 1 mM. In contrast, most purine nucleosides do not support sugar-free growth for one of the following reasons. The generation of ribose-1-P from nucleoside phosphorylase activity is necessary to provide all essential functions of sugar metabolism. Some nucleosides, e.g. xanthosine, did not support growth because they are poor substrates for this enzyme. De novo pyrimidine synthesis was inhibited greater than 80% by adenosine or high concentrations of inosine, e.g. 10 mM, which prevented growth on these nucleosides; in contrast, pyrimidine synthesis was inhibited only marginally on 1 mM inosine or guanosine, but normal growth was only seen on 1 mM inosine, not on guanosine. The inhibition of de novo adenine nucleotide synthesis prevented growth on guanosine, since guanine nucleotides could not be converted to adenine nucleotides. Guanine nucleotides were necessary for this inhibition of purine synthesis, since a mutant blocked in their synthesis grew normally on guanosine. De novo purine synthesis was severely inhibited by adenosine, inosine, or guanosine, but in contrast to guanosine, adenosine and inosine could provide all purine requirements by direct nucleotide conversions.

Comparative energy metabolism in cultured heart muscle and HeLa cells.

The yields of energy from oxidation of fatty acids, glucose, and glutamine were compared in cultures of chick embryo heart muscle (heart) and HeLa cells. Aerobic energy production, as measured by oxygen utilization, was comparable in the two cell types. In media containing dialyzed sera, the rates of incorporation of fatty acids directly into lipids were similar in both cells and accounted for greater than 97% of fatty acid metabolism in HeLa cells. However, in heart cells only 45% ended in lipid, 42% in protein, and 13% was released as CO2; the latter two products probably reflect the oxidation of fatty acids to acetyl-coenzyme A (-CoA) and its subsequent metabolism in the citrate cycle. Increased serum concentration in the medium did not affect fatty acid metabolism in HeLa cultures, but resulted in greater oxidation by heart cells (greater than 100 times that by HeLa cells). The metabolisms of both glucose and glutamine were similar in heart and HeLa cells with greater than or equal to 60% of glucose carbon ending as medium lactate and only 3-5% converted to acetyl-CoA. About 25% of glutamine carbon ended as CO2 and increased utilizations with increasing serum concentrations was accountable in both cells by increased lactate from glucose and glutamate from glutamine. CO2 production (and energy) from glutamine was independent of glutamine concentration within a tenfold range of physiological concentrations. The yields of energy have been calculated. In 10% dialyzed calf serum, oxidation of glutamine carbon provided about half of the total energy in heart cells; glucose about 35-45%, with most coming from glycolysis; oxidation of fatty acid carbon provided only 5-10%. That greater than 90% of the aerobic energy comes from glutamine in both cells can account for the comparable rates of oxygen utilization. HeLa cells derived little or no energy from fatty acids.