Inactivation of the murine pyruvate dehydrogenase (Pdha1) gene and its effect on early embryonic development.

A deficiency of pyruvate dehydrogenase complex (PDC) in humans results in lactic acidosis and neurological dysfunction that frequently results in death during infancy. Using gene targeting technology, a silent mutation was introduced into the murine X-linked Pdha1 gene that encodes the alpha subunit of the pyruvate dehydrogenase or E1 component of the complex. Two loxP sequences were introduced into intronic sequences flanking exon 8 to generate the Pdha1(flox8) allele. In vitro studies in embryonic stem cells demonstrated that deletion of exon 8 ablated PDC activity. Homozygous Pdha1(flox8) females were bred with male mice carrying a wild-type Pdha1 allele and a transgene that ubiquitously expresses the Cre recombinase to produce progeny with a deletion in exon 8, Pdha1(Deltaex8). The majority of progeny were found to be mosaic with the presence of both the flox and deleted alleles, and there were no apparent phenotypic effects associated with the null allele. The mosaic mice were interbred to increase the degree of mosaicism for the Pdha1(Deltaex8) allele in the subsequent generation, resulting in a significantly smaller litter size (54% reduction). Embryos carrying predominantly the Pdha1(Deltaex8) allele were found to be globally delayed in development by 9.5 days postcoitus, with resorption occurring over the following several days. These findings demonstrate an essential role for oxidative metabolism of glucose during the early postimplantation period of prenatal development.

Sterol regulatory element-binding protein-1c mimics the negative effect of insulin on phosphoenolpyruvate carboxykinase (GTP) gene transcription.

We have assessed the potential role of sterol regulatory element-binding protein-1c (SREBP-1c) on the transcription of the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (EC ) (PEPCK-C). SREBP-1c introduced into primary hepatocytes with an adenovirus vector caused a total loss of PEPCK-C mRNA and a marked induction of fatty acid synthase mRNA that directly coincided with the appearance of SREBP-1c in the hepatocytes. It also blocked the induction of PEPCK-C mRNA by cAMP and dexamethasone in these cells. In contrast, a dominant negative form of SREBP-1c (dnSREBP-1c) stimulated the accumulation of PEPCK-C mRNA in these cells. SREBP-1c completely blocked the induction of PEPCK-C gene transcription by the catalytic subunit of protein kinase A (PKA), and increasing concentrations of dnSREBP-1c reversed the negative effect of insulin on transcription from the PEPCK-C gene promoter in WT-IR cells. The more than 10-fold induction of PKA-stimulated PEPCK-C gene transcription caused by the co-activator CBP, was also blocked by SREBP-1c. In addition, dnSREBP-1c reversed the strong negative effect of E1A and NF1 on PKA-stimulated transcription from the PEPCK-C gene promoter. An analysis of the possible site of action of SREBP-1c using stepwise truncations of the PEPCK-C gene promoter indicated that the negative effect of SREBP-1c on transcription is exerted at a site between -355 and -277. We conclude that SREBP-1c is an intermediate in the action of insulin on PEPCK-C gene transcription in the liver and acts by blocking the stimulatory effect cAMP that is mediated via an interaction with cAMP-binding protein.

Biological properties of poly-L-lysine-DNA complexes generated by cooperative binding of the polycation.

We have evaluated the effect of NaCl concentration on the mode of binding of poly-L-lysine to DNA and the resulting structural and functional features of the condensed DNA particles using DNA precipitation, DNase I resistance, electron microscopy, and receptor-mediated gene transfer assays. At a high concentration of NaCl and in the presence of excess DNA, poly-L-lysine interacted with DNA cooperatively, fully condensing some of the DNA and leaving the rest of the DNA unbound. At low NaCl concentrations, poly-L-lysine molecules interacted with DNA in a noncooperative fashion, i.e. they bind randomly to the whole population of DNA molecules. Cooperative binding of poly-L-lysine to DNA occurred over a narrow range of NaCl concentrations, and the specific salt concentration depended on the length of the poly-L-lysine. The ability of condensed DNA to withstand digestion by DNase I was correlated with the structural features of the condensed DNA as determined by electron microscopy. Using our condensation procedure, cooperative binding of poly-L-lysine to DNA is a necessary prerequisite for the preparation of condensed DNA having a spherical shape and a diameter of 15-30 nm. Condensed DNA, containing galactosylated poly-L-lysine, was evaluated further for the extent and specificity of receptor-mediated gene transfer into HuH-7 human hepatoma cells via the asialoglycoprotein receptor. Efficient receptor-mediated transfection occurred only when condensed DNA complexes had a spherical shape with a diameter of 15-30 nm; asialofetuin, a natural ligand for the asialoglycoprotein receptor, inhibited this process by up to 90%. Our results support the importance of appropriate DNA condensation for the uptake and ultimate expression of DNA in hepatic cells.

Sodium butyrate induces transcription from the G alpha(i2) gene promoter through multiple Sp1 sites in the promoter and by activating the MEK-ERK signal transduction pathway.

Sodium butyrate, an erythroid differentiation inducer and a histone deacetylase inhibitor, increases G alpha(i2) levels in differentiating K562 cells. Here we show that sodium butyrate induces G alpha(i2) gene transcription via sequences at -50/-36 and -92/-85 in the G alpha(i2) gene promoter. Both sequences contain core sequence motif for Sp1 binding; electrophoretic mobility shift as well as supershift assays confirmed binding to Sp1. Transcription from the G alpha(i2) gene promoter was also activated by two other histone deacetylase inhibitors, trichostatin A and Helminthsporium carbonium toxin (HC toxin), which also induce erythroblastic differentiation in K562 cells. However, hydroxyurea, a potent erythroid differentiation inducer in these cells, did not activate transcription from this gene promoter, indicating that promoter activation is inducer-specific. Mutations within the Sp1 sites at -50/-36 and -92/-85 in the G alpha(i2) gene promoter substantially decreased transcriptional activation by sodium butyrate, trichostatin A, or HC toxin. Transfection with constitutively activated ERKs indicated that this promoter can be activated through the MEK-ERK signal transduction pathway. Inhibition of the MEK-ERK pathway with U0126 or reduction in the expression of endogenous ERK with an antisense oligonucleotide to ERK significantly inhibited sodium butyrate- and HC toxin-induced transcription but had no effect on trichostatin A-induced transcription. Inhibition of the JNK and p38 MAPKs, using selective inhibitors, had no effect on sodium butyrate-induced transcription. In cells in which sodium butyrate induction of promoter activation had been inhibited by various concentrations of U0126, constitutively activated ERK2 reversed this inhibition. These results show that the MEK-ERK signal transduction pathway is important in butyrate signaling, which eventually converges in the cell nucleus.

The use of transgenic mice to analyze the role of accessory factor two in the regulation of phosphoenolpyruvate carboxykinase (GTP) gene transcription during diabetes.

The regulation of transcription of the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (PEPCK-C) (4.1.1.32) during diabetes is a complex process that involves a number of regulatory elements in the PEPCK-C gene promoter. The accessory factor 2 (AF2)-binding region that is contained within the glucocorticoid regulatory unit of the PEPCK-C gene promoter (-451 to -353) has been implicated in the action of both insulin and glucocorticoids on PEPCK-C gene transcription. To determine the role of AF2 in these processes, we have generated a mouse model bearing a transgene that contains the PEPCK-C gene promoter with a mutation in the AF2-binding region. This promoter is linked to the structural gene for human growth hormone that is biologically inactive (AF2-2000/hGx). In the absence of the AF2 regulatory element, the transcription of the transgene in the liver is not induced by diabetes but is inhibited by the administration of insulin. There is also a marked reduction in the response of the AF2-2000/hGx gene in the kidney to the administration of glucocorticoids. The AF2-2000/hGx gene in the liver responds normally to a high carbohydrate diet with a marked decrease in gene transcription. This suggests that insulin is not exerting its usual negative effect on the PEPCK-C gene promoter through the AF2 site. In contrast, the response of this transgene to a high fat/carbohydrate-free diet is severely blunted. Our results support a role for the AF2 site in the PEPCK-C gene promoter in the effect of glucocorticoids, but not insulin, on PEPCK-C gene transcription in the liver.

Abdominal cross-sectional imaging for inpatients with abnormal liver function test results: yield and usefulness.

BACKGROUND: Abdominal cross-sectional imaging is often performed to evaluate abnormal liver function test (LFT) results in hospitalized patients. However, few data are available regarding the yield and usefulness of imaging inpatients for the indication of abnormal LFT results, the process of requesting abdominal imaging studies, or the response to their findings. METHODS: We retrospectively reviewed abdominal imaging scans that were obtained during a 27-month period. We matched the imaging studies done with the indication of abnormal LFT results; all scans were requested using computerized physician order entry. Reports were coded for interpretation and associated process step results. To determine the usefulness of the imaging studies, a random sample of patient charts with positively coded imaging studies were reviewed. Imaging examinations were considered useful if they provided new diagnostic information and/or changed subsequent patient care. RESULTS: Of 6494 abdominal imaging studies, 856 were performed for the indication of abnormal LFT results and matched to both image reports and laboratory results. Report coding judged 37% of interpretations as clinically significant, including 27% with "positive" (abnormal results and explain the abnormal LFT results) examinations. Among the positive examinations, the most common diagnoses were biliary obstruction (25%), cholecystitis (21%), malignancy (20%), and cirrhosis (14%). Positively coded reports provided new clinical information in 63% of these studies and changed patient care in 42% of cases. Process measures assessed provision of additional information to and from radiologists (69% and 8%, respectively) and the frequency with which the findings of current abdominal imaging studies were compared with those of prior studies (59%). CONCLUSION: Abdominal cross-sectional imaging studies performed on inpatients with abnormal LFT results had a high diagnostic yield and frequently changed patient care.

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.

Mice with a deletion in the gene for CCAAT/enhancer-binding protein beta have an attenuated response to cAMP and impaired carbohydrate metabolism.

Fifty percent of the mice homozygous for a deletion in the gene for CCAAT/enhancer-binding protein beta (C/EBP beta-/- mice; B phenotype) die within 1 to 2 h after birth of hypoglycemia. They do not mobilize their hepatic glycogen or induce the cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK). Administration of cAMP resulted in mobilization of glycogen, induction of PEPCK mRNA, and a normal blood glucose; these mice survived beyond 2 h postpartum. Adult C/EBP beta-/- mice (A phenotype) also had difficulty in maintaining blood glucose levels during starvation. Fasting these mice for 16 or 30 h resulted in lower levels of hepatic PEPCK mRNA, blood glucose, beta-hydroxybutyrate, blood urea nitrogen, and gluconeogenesis when compared with control mice. The concentration of hepatic cAMP in these mice was 50% of controls, but injection of theophylline, together with glucagon, resulted in a normal cAMP levels. Agonists (glucagon, epinephrine, and isoproterenol) and other effectors of activation of adenylyl cyclase were the same in liver membranes isolated from C/EBP beta-/- mice and littermates. The hepatic activity of cAMP-dependent protein kinase was 80% of wild type mice. There was a 79% increase in the concentration of RI alpha and 27% increase in RII alpha in the particulate fraction of the livers of C/EBP beta-/- mice relative to wild type mice, with no change in the catalytic subunit (C alpha). Thus, a 45% increase in hepatic cAMP (relative to the wild type) would be required in C/EBP beta-/- mice to activate protein kinase A by 50%. In addition, the total activity of phosphodiesterase in the livers of C/EBP beta-/- mice, as well as the concentration of mRNA for phosphodiesterase 3A (PDE3A) and PDE3B was approximately 25% higher than in control animals, suggesting accelerated degradation of cAMP. C/EBP beta influences the regulation of carbohydrate metabolism by altering the level of hepatic cAMP and the activity of protein kinase A.

Molecular and cellular adaptations to carbohydrate and fat intake.

Adaptation to carbohydrate and fat intake involves changes in a number of biochemical parameters at the cellular level. A change in the concentration of fat or carbohydrate in the blood acts directly to influence metabolic pathways by altering the flux of intermediates into cells. This in turn alters the concentration of hormones and other signaling molecules and changes the rate of expression of genes coding for key regulatory proteins or enzymes in metabolic pathways. These effects occur at different rates and in a tissue-specific manner in response to diet. A key metabolic adaptation involves changes in the level of expression of genes coding for proteins of critical importance in energy metabolism; this is largely due to an altered rate of transcription of selected genes under the control of hormones and/or carbohydrate and lipid. The mediators of this effect are transcription factors, that is, nuclear proteins which integrate the effects of hormones and substrates with the transcription process by binding to response elements in the promoters of regulated genes and interacting with the transcription machinery at the TATA box, thereby altering the activity of RNA polymerase II. In this review we will outline the hierarchy of cellular adaptations to diet and will emphasize the latest concepts of gene regulation in response to metabolites and hormones. In particular, we will review the role of the various transcription factors involved in the regulated expression of the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.132), a key gluconeogenic enzyme.

CREB binding protein coordinates the function of multiple transcription factors including nuclear factor I to regulate phosphoenolpyruvate carboxykinase (GTP) gene transcription.

Nuclear factor I (NFI) binds to a region of the phosphoenolpyruvate carboxykinase (GTP) (PEPCK) gene promoter adjacent to the cAMP regulatory element (CRE) and inhibits the induction of transcription from the gene promoter caused by the catalytic subunit of protein kinase A. In vivo footprinting studies demonstrated that both the CRE and the NFI-binding site are occupied by transcription factors, regardless of the presence of factors that stimulate (dibutyryl cAMP or dexamethasone) or inhibit (insulin) transcription from the PEPCK gene promoter. The NFI effects on transcription from the PEPCK gene promoter were observed even in the absence of the NFI binding site, suggesting the possibility of other weaker binding sites on the promoter or an interaction of NFI with a transcriptional co-activator. A mammalian two-hybrid system was used to demonstrate direct interaction between the transactivation domain of NFI-C and the CREB binding domain of the CREB-binding protein (CBP). Overexpression of a gene fragment encoding the CREB binding domain of CBP stimulates transcription from the PEPCK gene promoter. The inhibitory effect of NFI on transcription of the PEPCK gene induced by the catalytic subunit of protein kinase A appears to be the result of an interaction between NFI and the CREB-binding protein in which NFI competes with CREB for binding to the CREB-binding site on CBP. In contrast, glucocorticoids and thyroid hormone use the steroid hormone receptor binding domain of CBP to stimulate transcription from the PEPCK gene promoter. NFI-A combines with dexamethasone or thyroid hormone in an additive manner to stimulate PEPCK gene transcription. We conclude that CBP coordinates the action of the multiple factors known to control transcription of the PEPCK gene.

Hypoglycemia and impaired hepatic glucose production in mice with a deletion of the C/EBPbeta gene.

The transcription factor CCAAT/enhancer-binding protein beta (C/EBPbeta) is enriched in liver and adipose tissue and controls the expression of a wide variety of genes coding for important metabolic pathways, including gluconeogenesis and lipid synthesis. To investigate the role of C/EBPbeta on glucose homeostasis, we studied mice with a targeted deletion of the gene for C/EBPbeta-/- mice. Adult C/EBPbeta-/- mice have hypoglycemia after an 18-hour fast, accompanied by lower hepatic glucose production (40% of that of wild-type mice), with no change in plasma insulin and a lower concentration of plasma free fatty acids (FFA). Glucagon infusion during a pancreatic clamp acutely stimulated hepatic glucose production by 38% in wild-type animals, with no change detected in C/EBPbeta-/- mice. Unexpectedly, both the basal and glucagon-stimulated hepatic cyclic adenosine monophosphate (cAMP) levels were lower in C/EBPbeta-/- mice, indicating an essential role for C/EBPbeta in controlling proximal signal transduction. Fasting hypoglycemia was associated with normal levels of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) gene expression, however net liver glycogenolysis was impaired in C/EBPbeta-/- mice. FFA release from isolated adipose tissue in response to epinephrine was 68% lower in C/EBPbeta-/- mice than in control animals; however, N6,O2'-dibutyryladenosine (Bt2) cAMP stimulated a twofold increase in FFA release in C/EBPbeta-/- compared with no further increase in wild-type mice. Because a deletion in the gene for C/EBPbeta reduces blood glucose and circulating FFA, it could be an important therapeutic target for the treatment of non-insulin-dependent diabetes and possibly obesity, based on designing antagonists that decrease C/EBPbeta activity.

Glycemia-lowering effect of cobalt chloride in the diabetic rat: role of decreased gluconeogenesis.

Results of previous studies indicated that treatment of diabetic rats (induced by streptozotocin) with cobalt chloride (CoCl2) resulted in a significant decrement in serum glucose concentration. The present study was designed to determine the potential role of enhanced glucose uptake vs. decreased glucose production in the above response. The rate of systemic appearance of glucose, measured under fasting conditions using [3-3H]glucose tracer, was reduced from 35.5 +/- 2.5 to 17.5 +/- 1.8 micromol . kg-1 . min-1 in diabetic rats treated with 2 mM CoCl2 added to the drinking water for 10-14 days (P < 0.01). Tissue accumulation of intravenously administered 2-deoxy-[14C]glucose was significantly reduced in kidney and eye of diabetic rats treated with CoCl2, whereas the uptake remained unchanged in several other tissues including cerebrum, red and white skeletal muscle, heart, and liver. The relative content of phosphoenolpyruvate carboxykinase (PEPCK) mRNA was increased 3.1-fold in livers of diabetic compared with normal rats (P < 0.001), and treatment of diabetic rats with CoCl2 decreased hepatic PEPCK mRNA levels to normal. The content of PEPCK mRNA in the liver was decreased by 33% in CoCl2-treated normal rats (P < 0.05). Treatment with CoCl2 resulted in no change in cAMP levels in the livers of either diabetic or normal rats. These results suggest that the glycemia-lowering effect of CoCl2 is mediated by reductions in the rate of systemic appearance of glucose and hepatic gluconeogenesis.

Nuclear factor I regulates expression of the gene for phosphoenolpyruvate carboxykinase (GTP).

Nuclear factor-I (NFI) binds to the phosphoenolpyruvate carboxykinase (GTP) (PEPCK) gene promoter immediately 5' to the cAMP regulatory element (CRE). This suggests an interaction between NFI and factors that bind the CRE. Of the four NFI isoforms expressed in mammalian tissues, NFI-A and -B stimulate basal transcription from the PEPCK gene promoter in HepG2 cells, while NFI-C and -X are slightly inhibitory. All four NFI isoforms abrogate the 20-fold protein kinase Ac (PKAc)-mediated induction of transcription from the PEPCK gene promoter. Normal PKAc-mediated induction was noted when the CRE was moved 10 base pairs 3' of its original location. However if the CRE was moved 5 base pairs 3', placing it out of phase with the other elements in the promoter, or moved 5' to -285 (the P3(I) site in the promoter), some PKA-mediated stimulation was lost. The NFI-C isoform effectively inhibited PKAc induction regardless of the relative positions of the CRE and the NFI binding sites. NFI-C also abrogated cAMP regulatory element-binding protein (CREB)-induced activity of wild type and mutant PEPCK promoters. There was some cooperativity in the binding of CREB and NFI to their respective binding sites but this did not appear to be physiologically important.

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.

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.

Biochemical and functional characterization of DNA complexes capable of targeting genes to hepatocytes via the asialoglycoprotein receptor.

Electrostatic binding of polycations or basic polypeptides to the DNA phosphate backbone has been previously described as a one-step process which results in uncontrolled aggregation and precipitation of the DNA in solution. We describe here a multistep process in which the condensation of DNA in the presence of poly-L-lysine can be controlled to produce particles of discrete size and shape suitable for receptor-mediated gene transfer in vivo and in vitro. The first step in this process involves the gradual accretion of poly-L-lysine onto the DNA phosphate backbone, until charges are neutralized. The addition of poly-L-lysine to a concentrated solution of DNA in this fashion prevents intermolecular aggregation of the DNA, presumably by promoting the formation of a nucleus of condensation along the length of each DNA molecule. The second stage of the process involves adjusting the ionic strength of the solvent to facilitate the solubilization of compact DNA.poly-L-lysine complexes. Several physical and biochemical parameters have been studied and correlated with the efficacy of DNA/ligand-poly-L-lysine particles in transferring genes to the liver of adult animals by receptor-mediated endocytosis.

The promoter regulatory regions of the genes for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) from the chicken and the rat have different species-specific roles in gluconeogenesis.

Hepatic expression of the gene for phosphoenolpyruvate carboxykinase (GTP) (PEPCK-C) (EC 4.1.1.32) in birds occurs prior to birth and decreases to negligible levels before hatching, whereas in mammals the gene for PEPCK-C in the liver is expressed at birth and is active throughout the life of the animal. The administration of cyclic AMP to adult chickens results in the induction of transcription of the gene for PEPCK-C and the transient accumulation of PEPCK-C mRNA in the liver. DNase I footprint analysis of 330 bp of the avian PEPCK-C promoter immediately 5' of the start-site of transcription indicated the presence of several protein binding domains, purified CAAT/enhancer binding protein alpha, cAMP regulatory element binding protein and nuclear factor-1 bound to these regions of the promoter. Sequences corresponding to an hepatic nuclear factor-1 binding domain and to the insulin response sequence, previously identified in the rat PEPCK-C promoter, were also found in the chicken PEPCK-C promoter. Co-transfection of an expression vector for CAAT/enhancer binding protein alpha or CAAT/enhancer binding protein beta markedly stimulated transcription from both the chicken and rat PEPCK-C promoters in human hepatoma cells. Sequences involved in the regulation of gene transcription by cyclic AMP and insulin were found to reside between -210 and +1 of the avian PEPCK-C promoter. In general, transcription from the avian promoter was more sensitive to inhibition by insulin than was noted for the rat PEPCK-C promoter, which may explain in part the lack of expression of the gene for PEPCK-C in the livers of adult birds.