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L Reshef

Publications and source records attributed to L Reshef.

At least 19 recordsLinked to original sources
PubMed2001

Glyceroneogenesis and the source of glycerol for hepatic triacylglycerol synthesis in humans.

Glyceroneogenesis, i.e. the synthesis of the glycerol moiety of triacylglycerol from pyruvate, has been suggested to be quantitatively important in both the liver and adipose tissue during fasting. However, the actual contribution of glyceroneogenesis to triacylglycerol synthesis has not been quantified in vivo in human studies. In the present study we have measured the contribution of glycerol and pyruvate to in vivo synthesis of hepatic triacylglycerol in nonpregnant and pregnant women after an overnight fast. Five nonpregnant women were administered [(13)C(3)]glycerol tracer as prime constant rate infusion, and the appearance of tracer in plasma glucose and triacylglycerol was quantified using gas chromatography-mass spectrometry. The contribution of pyruvate to hepatic triacylglycerol was quantified in nonpregnant and pregnant women using the deuterium labeling of body water method. The appearance of [(2)H] in hydrogens on C(1) and C(3) of triacylglycerol was measured following periodate oxidation of the glycerol isolated from hydrolyzed triacylglycerol. After a 16-h fast, approximately 6.1% of the plasma triacylglycerol pool was derived from plasma glycerol, whereas 10 to 60% was derived from pyruvate in nonpregnant women and pregnant women early in gestation. Our data suggest that glyceroneogenesis from pyruvate is quantitatively a major contributor to plasma triacylglycerol synthesis and may be important for the regulation of very low density lipoprotein triacylglycerol production. Our data also suggest that 3-glycerol phosphate is in rapid equilibrium with the triosephosphate pool, resulting in rapid labeling of the triose pool by the administered tracer glycerol. Because the rate of flux of triosephosphate to glucose during fasting far exceeds that to triacylglycerol, more glycerol ends up in glucose than in triacylglycerol. Alternatively, there may be two distinct pools of 3-glycerol phosphate in the liver, one involved in generating triosephosphate from glycerol and the other involved in glyceride-glycerol synthesis.

Blood Glucose↗
PubMed1999

Repression and activation of transcription of phosphoenolpyruvate carboxykinase gene during liver development.

Transcriptional activation of the hepatic phosphoenolpyruvate carboxykinase (PEPCK) gene at birth is critical since PEPCK appearance initiates hepatic gluconeogenesis. A delayed appearance results in hypoglycemia, while a premature appearance results in neonatal diabetes, both are incompatible with sustaining life. Experiments using transgenic mice and transfected hepatoma cells suggest that both repression and activation underlie the correct onset of hepatic PEPCK gene transcription. In transgenic mice, transgenes driven by the proximal PEPCK promoter are prematurely expressed in the fetal liver and over-expressed in the neonatal liver, indicating that sequences upstream of the proximal promoter restrain perinatal expression. In Hepa1c1c7 cells, which mimic the fetal liver, the proximal PEPCK promoter (597 bp) exhibited a 3. 5-10-fold higher activity than longer promoters. Repression of the longer promoter (2000 bp) was diminished upon deletion of the sequence spanning positions(-840) to(- 1116) which contains a PPAR/RXR recognition element. The intact 2000 bp PEPCK promoter could be markedly activated by co-transfecting the transcription factor HNF-1 together with C/EBP. It could be repressed by co-transfection with RXRalpha and adding PPARalpha relieved this inhibition.

Animals↗
PubMed1997

Role of the isoforms of CCAAT/enhancer-binding protein in the initiation of phosphoenolpyruvate carboxykinase (GTP) gene transcription at birth.

The gene for phosphoenolpyruvate carboxykinase (PEPCK), a target of CCAAT/enhancer-binding protein-alpha (C/EBPalpha) and -beta (C/EBPbeta), begins to be expressed in the liver at birth. Mice homozygous for a deletion in the gene for CEBPalpha (C/EBPalpha-/- mice) die shortly after birth of hypoglycemia, with no detectable hepatic PEPCK mRNA and negligible hepatic glycogen stores. Half of the mice homozygous for a deletion in the gene for CEBPbeta (C/EBPbeta-/- mice) have normal glucose homeostasis (phenotype A), and the other half die at birth of hypoglycemia due to a failure to express the gene for PEPCK and to mobilize hepatic glycogen (phenotype B). Insulin deficiency induces C/EBPalpha and PEPCK gene transcription in the livers of 19-day fetal rats, whereas dibutyryl cyclic AMP (Bt2cAMP) increases the expression of the gene for C/EBPbeta and causes a transient burst of PEPCK mRNA. Bt2cAMP induces PEPCK mRNA in the livers of fetal C/EBPalpha-/- mice, but at only 20% of the level of control animals; however, there is no induction of PEPCK mRNA if the cyclic nucleotide is injected into C/EBPalpha-/- mice immediately after delivery. The expression of the gene for C/EBPbeta is markedly induced in the livers of C/EBPalpha-/- mice within 2 h after the administration of Bt2cAMP. C/EBPbeta-/- mice injected at 20 days of fetal life with Bt2cAMP have a normal pattern of induction of hepatic PEPCK mRNA. In C/EBPbeta-/- mice with phenotype B, the administration of Bt2cAMP immediately after delivery induces PEPCK mRNA, causes the mobilization of hepatic glycogen, and maintains normal glucose homeostasis for up to 4 h (duration of the experiment). We conclude that C/EBPalpha is required for the cAMP induction of PEPCK gene expression in the liver and that C/EBPbeta can compensate for the loss of C/EBPalpha if its concentration is induced to appropriate levels.

Animals↗
PubMed1997

Involvement of HNF-1 in the regulation of phosphoenolpyruvate carboxykinase gene expression in the kidney.

The cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (PEPCK) gene is differentially expressed in several tissues. A specific set of regulatory elements in the promoter are responsible for the control of PEPCK gene transcription and, in turn, determine its distinct metabolic role in each tissue. DNase I footprinting analysis of the PEPCK promoter, using nuclear proteins from tissues which express the gene for PEPCK, and transient expression assays in renal cell lines have demonstrated that the HNF-1 recognition motif (P2) in the PEPCK promoter characterizes kidney-specific expression. This site is required also for the response to acidosis. Since the P2 site is not involved in the expression of the PEPCK gene in the liver, we propose that its critical role in the kidney stems from a combination of abundance of HNF-1 together with low concentrations of members of the C/EBP family in this tissue.

Acids↗
PubMed1997

Regulation of phosphoenolpyruvate carboxykinase (GTP) gene expression.

Phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) is a key enzyme in the synthesis of glucose in the liver and kidney and of glyceride-glycerol in white adipose tissue and the small intestine. The gene for the cytosolic form of PEPCK (PEPCK-C) is acutely regulated by a variety of dietary and hormonal signals, which result in alteration of synthesis of the enzyme. Major factors that increase PEPCK-C gene expression include cyclic AMP, glucocorticoids, and thyroid hormone, whereas insulin inhibits this process. PEPCK-C is absent in fetal liver but appears at birth, concomitant with the capacity for gluconeogenesis. Regulatory elements that control transcription of the PEPCK-C gene in liver, kidney, and adipose tissue have been delineated, and many of the transcription factors that bind to these elements have been identified. Transgenic mice have been especially useful in elucidating the physiological roles of specific sequence elements in the PEPCK-C gene promoter and in demonstrating the key role played at these sites by the isoforms of CAAT/enhancer binding protein in patterning of PEPCK-C gene expression during the perinatal period. The PEPCK-C gene provides a model for the metabolic control of gene transcription.

Animals↗
PubMed1996

Identification of differentially expressed genes during hepatocytes development and characterization of their prenatal hormonal induction.

Upon birth, the liver acquires new functions as a result of the initiation of expression of key enzymes. One example is the initiation of gluconeogenesis which depends on the induced appearance of phosphoenolpyruvate carboxykinase (P-pyruvate-CK) at birth. To characterize other genes that undergo such regulation, a differential screening was performed on a cDNA library from well-differentiated hepatoma cells. The pattern of tissue-specific and developmental-specific expression was determined for seven genes. Three clones, out of which two encode for the known genes alcohol dehydrogenase class I (ADH) and phenylalanine 4-monooxygenase (PAH) and a new gene (clone 116-3), exhibited a pattern of expression similar to that of the P-pyruvate-CK gene, i.e. their expression was liver and kidney specific and induced in the liver upon birth. Determination of the sequence of clone 116-3 revealed that it belonged to the UDP-glucuronosyltransferases type 2 (UGT2) family and thus was named UGT2B-rH4. To examine whether expression of the various genes could be prematurely induced by hormones in the fetal liver, either high levels of cAMP or low levels of insulin were induced in utero. The results demonstrated that cAMP induced a marked expression only of the genes for P-pyruvate-CK and ADH but not of those for PAH or UGT2B-rH4, while insulin deficiency induced premature expression of all four genes. We suggest that a set of genes whose expression is specifically induced in the liver upon birth can be prematurely induced by the hormones in utero.

Alcohol Dehydrogenase↗
PubMed1995

Cooperation between transcription factors regulates liver development.

Characterization and cloning of liver-enriched transcription factors have provided the tools to study the regulation of liver differentiation. Characterization of the temporal and spatial expression of these factors have shown a sequential order of appearance, in coordination with the expression of their target genes, during liver development. Evidence has accumulated showing cooperation between distinct factors in regulating liver-specific gene expression. Since each of these factors is not uniquely expressed in the liver, yet, the liver is the only tissue that expresses all of these factors, the cooperation between the coexisting liver-enriched factors could constitute the basis for the regulation of liver-specific gene expression.

Animals↗
PubMed1994

Transcriptional regulation of the phosphoenolpyruvate carboxykinase gene by cooperation between hepatic nuclear factors.

To study the transcriptional regulation of the liver gluconeogenic phenotype, the underdifferentiated mouse Hepa-1c1c7 (Hepa) hepatoma cell line was used. These cells mimicked the fetal liver by appreciably expressing the alpha-fetoprotein and albumin genes but not the phosphoenolpyruvate carboxykinase (PEPCK) gene. Unlike the fetal liver, however, Hepa cells failed to express the early-expressed factors hepatocyte nuclear factor 1 alpha (HNF-1 alpha) and HNF-4 and the late-expressed factor C/EBP alpha, thereby providing a suitable system for examining possible cooperation between these factors in the transcriptional regulation of the PEPCK gene. Transient transfection assays of a chimeric PEPCK-chloramphenicol acetyltransferase construct showed a residual PEPCK promoter activity in the Hepa cell line, which was slightly stimulated by cotransfection with a single transcription factor from either the C/EBP family or HNF-1 alpha but not at all affected by cotransfection of HNF-4. In contrast, cotransfection of the PEPCK construct with members from the C/EBP family plus HNF-1 alpha resulted in a synergistic stimulation of the PEPCK promoter activity. This synergistic effect depended on the presence in the PEPCK promoter region of the HNF-1 recognition sequence and on the presence of two C/EBP recognition sequences. The results demonstrate a requirement for coexistence and cooperation between early and late liver-enriched transcription factors in the transcriptional regulation of the PEPCK gene. In addition, the results suggest redundancy between members of the C/EBP family of transcription factors in the regulation of PEPCK gene expression.

Animals↗
PubMed1992

Differential regulation of the rat phosphoenolpyruvate carboxykinase gene expression in several tissues of transgenic mice.

The selective expression of a unique copy gene in several mammalian tissues has been approached by studying the regulatory sequences needed to control expression of the rat phosphoenolpyruvate carboxykinase (PEPCK) gene in transgenic mice. A transgene containing the entire PEPCK gene, including 2.2 kb of the 5'-flanking region and 0.5 kb of the 3'-flanking region, exhibits tissue-specific expression in the liver, kidney, and adipose tissue, as well as the hormonal and developmental regulation inherent to endogenous gene expression. Deletions of the 5'-flanking region of the gene have shown the need for sequences downstream of position -540 of the PEPCK gene for expression in the liver and sequences downstream of position -362 for expression in the kidney. Additional sequences upstream of position -540 (up to -2200) are required for expression in adipose tissue. In addition, the region containing the glucocorticoid-responsive elements of the gene used by the kidney was identified. This same sequence was found to be needed specifically for developmental regulation of gene expression in the kidney and, together with upstream sequences, in the intestine. The apparently distinct sequence requirements in the various tissues indicate that the tissues use different mechanisms for expression of the same gene.

Aging↗
PubMed1991

Cis-regulatory elements that confer differential expression upon the rat gene encoding phosphoenolpyruvate carboxykinase in kidney and liver.

The PCK gene, encoding cytosolic phosphoenolpyruvate carboxykinase, is specifically expressed in gluconeogenic tissues, liver and kidney. Hence it serves as a model of a class of single-copy genes whose transcription is restricted to a few tissues, rather than a unique tissue. To begin delineating the mechanisms that govern this pattern of expression, cis-regulatory elements of PCK were examined using transient transfection assays in PCK-expressing kidney and hepatoma cell lines. The analyses enabled us to identify a proximal element, between nucleotide (nt) positions -121 and -98, relative to the transcription start point that is sufficient for specific expression in kidney cells, but is just one of the elements required for expression in hepatoma cells. A distal element (between nt -487 and -417), which is essential for hepatoma-specific expression, is not needed in kidney cells. We suggest that the differential regulation of PCK expression in the liver and kidney results from an interplay between different cis-regulatory elements and trans-acting factors.

Animals↗
PubMed1991

Regulation of tissue- and development-specific gene expression in the liver.

The liver is equipped with a repertoire of enzymatic activities essential for executing its specialized role in metabolism, the expression of which is regulated during development. The liver-specific phenotype is the consequence of a developmental tissue-specific program of gene expression. Sequences close to many characterized structural liver-specific genes (cis-regulatory elements) regulate their transcription. Identification of such cis-regulatory elements, capable of conferring a hepatocyte-specific gene expression, has been achieved by the introduction of chimeric genes into germ lines, producing transgenic animals, into differentiated cultured cells and into a cell-free transcription system. Such cis-elements in the DNA are recognized by specific DNA-binding nuclear proteins (trans-acting factors) which are liver-enriched and developmentally controlled. The interaction of defined cis-acting elements, near liver-specific genes, with liver-specific trans-acting factors might result in the differentiation of cells of the endoderm lineage into hepatocyte cells.

Animals↗
PubMed1990

Developmentally regulated interactions of liver nuclear factors with the rat phosphoenolpyruvate carboxykinase promoter.

A sequential pattern of interactions of trans-acting factors in rat liver with the phosphoenolpyruvate carboxykinase promoter during late development was observed. A liver-enriched factor, possibly AF1, interacted with the promoter in fetal liver, whereas a factor with the characteristics of C/EBP bound the promoter after birth with the onset of the gene expression.

Age Factors↗
PubMed1989

Separate cis-regulatory elements confer expression of phosphoenolpyruvate carboxykinase (GTP) gene in different cell lines.

The gene encoding cytosolic phosphoenolpyruvate carboxykinase (GTP) [PEPCK; GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32], a key enzyme in gluconeogenesis and glyceroneogenesis, is expressed in tissues that arise from different embryonal origins: the gluconeogenic liver arises from endoderm, whereas the gluconeogenic kidney cortex and glyceroneogenic adipose tissue arise from the mesoderm. To identify the cis-regulatory elements conferring the differential gene expression, PEPCK chimeric genes were transfected into two rat hepatoma cell lines (H4IIEC3 and HTC-M1.1) and mouse adipocytes (3T3F442A), which express the endogenous gene, and into myoblasts and preadipocytes, which do not express it. The results demonstrate that 597 base pairs of the 5' flanking region of the PEPCK gene are sufficient to confer cell-specific gene expression in the PEPCK-expressing hepatoma cells and adipocytes. However, different elements within this 597-base-pair region enhance the gene expression in the hepatoma cells (endoderm) and adipocytes (mesoderm). In the hepatocytes, expression is conferred by two elements--one 5' of position -362 and the other 3' of position -98 with respect to the transcription start site. The region in between these two elements (from -362 to -98), which seems to inhibit the gene expression in the hepatocytes, confers enhanced expression in the adipocytes. Moreover, the distal positive regulatory element of the hepatocytes seems to be orientation and PEPCK promoter dependent. In contrast, the positive regulatory element of the adipocytes seems to act as a more typical enhancer. These results suggest that separate cis-regulatory elements confer cell-specific expression of the PEPCK gene.

Animals↗
PubMed1989

trans activation of rat phosphoenolpyruvate carboxykinase (GTP) gene expression by micro-coinjection of rat liver mRNA in Xenopus laevis oocytes.

To study the liver-specific trans activation of the rat phosphoenolpyruvate carboxykinase (PEPCK) gene, the PEPCK promoter was linked to a reporter gene and was microinjected into Xenopus laevis oocytes alone or in conjunction with rat liver poly(A)+ RNA. The rat liver mRNA markedly enhanced the expression of the PEPCK-chimeric construct. This effect appeared to be sequence specific, as it was dependent on the presence of the intact promoter. Moreover, the RNA effect was limited to mRNA preparations from PEPCK-expressing tissues only. Finally, microinjection of size-fractionated liver mRNA revealed that the trans-acting factor(s) is encoded by RNA of 1,600 to 2,000 nucleotides, providing a direct bioassay for the gene(s) involved in this tissue-specific trans-activation process.

Animals↗
PubMed1987

Fate of polyoma origin of replication after its direct introduction into mice.

Recently we have developed a method for direct introduction of calcium phosphate-precipitated DNA into newborn rats. To examine whether the foreign DNA can replicate, a plasmid containing a polyoma origin of replication was injected into newborn mice. The plasmid was found intact in liver and spleen and able to transform bacteria. The foreign DNA had disappeared by the seventh day after injection. Yet, the plasmid DNA containing the polyoma origin of replication had undergone replication in both the liver and the spleen.

Animals↗
PubMed1987

Glucocorticoids control phosphoenolpyruvate carboxykinase gene expression in a tissue specific manner.

Cytosolic Phosphoenolpyruvate carboxykinase is a key gluconeogenic enzyme which is expressed in a tissue specific manner in the liver, kidney and adipose tissue and is under hormonal control. The effect of glucocorticoids on expression of the gene coding for phosphoenolpyruvate carboxykinase in adipose tissue has been studied in vivo in rats and in vitro in adipose tissue organ culture and mouse 3T3 L1 adipocytes. Glucocorticoids, both in vivo and in vitro, repress the steady state level of phosphoenolpyruvate carboxykinase mRNA in the adipose tissue while increasing it in the kidney. The size of the mRNA and its 5' end are identical in adipose tissue and kidney, thus the same promoter is used in all tissues. The inhibitory effect of glucocorticoids on phosphoenolpyruvate carboxykinase gene expression was located at the level of transcription. As glucocorticoids are known to stimulate transcription of phosphoenolpyruvate carboxykinase gene in the liver and kidney, the inhibitory effect on its transcription in adipose tissue suggests that tissue specific transcription factors may modulate the effect of glucocorticoids.

Adipose Tissue↗
PubMed1987

Developmental acquisition of DNase I sensitivity of the phosphoenolpyruvate carboxykinase (GTP) gene in rat liver.

The sensitivity to DNase I digestion of the gene encoding rat phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) was assessed during development and prior to the onset of expression. This gene is resistant to DNase I digestion in nuclei isolated from livers of 19-day rat fetuses. Gradual acquisition of sensitivity of the phosphoenolpyruvate carboxykinase gene, which starts later than the 19th day of gestation and is completed by the 21st day, occurs before initiation of gene expression. As transcription of the phosphoenolpyruvate carboxykinase gene is not detected until birth, the events observed may represent a shift from a dormant to an active gene. Injection of N6,O2-dibutyryladenosine 3',5'-cyclic monophosphate into fetuses on the 19th day of gestation induces gene expression and sensitivity to DNase I digestion within 3 hr of treatment. While this short treatment does not affect the methylation pattern of the gene, longer treatment of fetuses (2 days) with dibutyryl-cAMP results in premature hypomethylation of the gene. A hierarchy of modifications of the phosphoenolpyruvate carboxykinase gene during development is discussed.

Animals↗