Hypothalamic melanin-concentrating hormone and estrogen-induced weight loss.

Melanin-concentrating hormone (MCH) is an orexigenic neuropeptide produced by neurons of the lateral hypothalamic area (LHA). Because genetic MCH deficiency induces hypophagia and loss of body fat, we hypothesized that MCH neurons may represent a specific LHA pathway that, when inhibited, contributes to the pathogenesis of certain anorexia syndromes. To test this hypothesis, we measured behavioral, hormonal, and hypothalamic neuropeptide responses in two models of hyperestrogenemia in male rats, a highly reproducible anorexia paradigm. Whereas estrogen-induced weight loss engaged multiple systems that normally favor recovery of lost weight, the expected increase of MCH mRNA expression induced by energy restriction was selectively and completely abolished. These findings identify MCH neurons as specific targets of estrogen action and suggest that inhibition of these neurons may contribute to the hypophagic effect of estrogen.

Effects of streptozotocin-induced diabetes and insulin treatment on the hypothalamic melanocortin system and muscle uncoupling protein 3 expression in rats.

Hypothalamic melanocortins are among several neuropeptides strongly implicated in the control of food intake. Agonists for melanocortin 4 (MC-4) receptors such as alpha-melanocyte-stimulating hormone (alpha-MSH), a product of proopiomelanocortin (POMC), reduce food intake, whereas hypothalamic agouti-related protein (AgRP) is a MC-4 receptor antagonist that increases food intake. To investigate whether reduced melanocortin signaling contributes to hyperphagia induced by uncontrolled diabetes, male Sprague-Dawley rats were studied 7 days after administration of streptozotocin (STZ) or vehicle. In addition, we wished to determine the effect of diabetes on muscle uncoupling protein 3 (UCP-3), a potential regulator of muscle energy metabolism. STZ diabetic rats were markedly hyperglycemic (31.3 +/- 1.0 mmol/l; P < 0.005) compared with nondiabetic controls (9.3 +/- 0.2 mmol/l). Insulin treatment partially corrected the hyperglycemia (18.8 +/- 2.5 mol/l; P < 0.005). Plasma leptin was markedly reduced in STZ diabetic rats (0.4 +/- 0.1 ng/ml; P < 0.005) compared with controls (3.0 +/- 0.4 ng/ml), an effect that was also partially reversed by insulin treatment (1.8 +/- 0.3 ng/ml). Untreated diabetic rats were hyperphagic, consuming 40% more food (48 +/- 1 g/day; P < 0.005) than controls (34 +/- 1 g/day). Hyperphagia was prevented by insulin treatment (32 +/- 2 g/day). In untreated diabetic rats, hypothalamic POMC mRNA expression (measured by in situ hybridization) was reduced by 80% (P < 0.005), whereas AgRP mRNA levels were increased by 60% (P < 0.01), suggesting a marked decrease of hypothalamic melanocortin signaling. The change in POMC, but not in AgRP, mRNA levels was partially reversed by insulin treatment. By comparison, the effects of diabetes to increase hypothalamic neuropeptide Y (NPY) expression and to decrease corticotropin-releasing hormone (CRH) expression were normalized by insulin treatment, whereas the expression of mRNA encoding the long form of the leptin receptor in the arcuate nucleus was unaltered by diabetes or insulin treatment. UCP-3 mRNA expression in gastrocnemius muscle from diabetic rats was increased fourfold (P < 0.005), and the increase was prevented by insulin treatment. The effect of uncontrolled diabetes to decrease POMC, while increasing AgRP gene expression, suggests that reduced hypothalamic melanocortin signaling, along with increased NPY and decreased CRH signaling, could contribute to diabetic hyperphagia. These responses, in concert with increased muscle UCP-3 expression, may also contribute to the catabolic effects of uncontrolled diabetes on fuel metabolism in peripheral tissues.

Leptin sensitive neurons in the hypothalamus.

A major paradigm in the field of obesity research is the existence of an adipose tissue-brain endocrine axis for the regulation of body weight. Leptin, the peptide mediator of this axis, is secreted by adipose cells. It lowers food intake and body weight by acting in the hypothalamus, a region expressing an abundance of leptin receptors and a variety of neuropeptides that influence food intake and energy balance. Among the most promising candidates for leptin-sensitive cells in the hypothalamus are arcuate nucleus neurons that co-express the anabolic neuropeptides, neuropeptide Y (NPY) and agouti-related peptide (AGRP), and those that express proopiomelanocortin (POMC), the precursor of the catabolic peptide, alphaMSH. These cell types contain mRNA encoding leptin receptors and show changes in neuropeptide gene expression in response to changes in food intake and circulating leptin levels. Decreased leptin signaling in the arcuate nucleus is hypothesized to increase the expression of NPY and AGRP. Levels of leptin receptor mRNA and leptin binding are increased in the arcuate nucleus during fasting, principally in NPY/AGRP neurons. These findings suggest that changes in leptin receptor expression in the arcuate nucleus are inversely associated with changes in leptin signaling, and that the arcuate nucleus is an important target of leptin action in the brain.

Kinetic analysis of human deoxycytidine kinase with the true phosphate donor uridine triphosphate.

Deoxycytidine kinase is the rate-limiting process in the activation for several clinically important antitumor agents. Previous studies have focused on deoxycytidine (dCyd) and adenosine triphosphate (ATP) as substrates for this enzyme. In view of recent data indicating that uridine triphosphate (UTP) is the physiologic phosphate donor for this enzyme, a study of the kinetic properties of dCyd kinase with dCyd and UTP was undertaken. The results presented here demonstrate that UTP and ATP produce kinetically distinguishable differences in nucleoside phosphorylation by dCyd kinase. At high dCyd concentrations, dCyd kinase exhibited substrate activation with ATP. In contrast, in the presence of UTP, substrate inhibition was observed at concentrations of dCyd greater than 3 microM. Inhibition by dCyd was noncompetitive with respect to UTP and could not be reversed by a 200-fold increase in UTP concentration, indicating that the inhibition was not due to dCyd binding at the nucleotide binding site. The kinetic mechanism for dCyd kinase was determined with dCyd and UTP as substrates. UTP was the preferred phosphate donor with a true Km value of 1 microM compared to 54 microM with ATP, resulting in a 50-fold greater substrate efficiency for UTP. Although the double-reciprocal plots with UTP produced parallel lines, initial velocity plots with other phosphate donors and product inhibition studies indicated that dCyd kinase formed a ternary complex with its substrates. The parallel lines with UTP were apparently due to a low dissociation constant for UTP, which was calculated as more than 13-fold lower than its Km value. Analysis of product inhibition studies indicated that dCyd kinase followed an ordered A-B random P-Q reaction sequence, with UTP as the first substrate to bind. In contrast, previous results demonstrated a random bi-bi sequence for dCyd kinase in the presence of ATP. The combined results indicate that the enzyme can follow a random bi-bi reaction sequence, but with UTP as the phosphate donor, the addition of nucleotide prior to dCyd is strongly preferred. The noncompetitive substrate inhibition, which was independent of UTP concentration, indicates that high concentrations of dCyd promote addition of the nucleoside prior to UTP, resulting in a lower velocity.

Delayed radiosensitization of human colon carcinoma cells after a brief exposure to 2',2'-difluoro-2'-deoxycytidine (Gemcitabine).

We have shown that 2',2'-difluoro-2'-deoxycytidine (dFdCyd; Gemcitabine), a deoxycytidine analogue, is a potent radiation sensitizer when cells are exposed to it continuously for >16 h in low concentrations (in the range of 10 nM). However, the most common method of clinical administration is by short-term infusion (30-90 min). Therefore, we wished to determine under what conditions dFdCyd could produce radiosensitization after a relatively brief exposure to drug. We hypothesized that the long half-life of the phosphorylated metabolites of dFdCyd would produce long-lasting dNTP pool perturbation, particularly dATP pools, leading to radiosensitization hours or even days after the drug was removed from the medium. We tested this hypothesis by exposing HT29 human colon cancer cells for 2 h to clinically relevant concentrations of dFdCyd, removing the drug from the medium, and assessing radiation sensitivity up to 72 h later. We found that 100 nM dFdCyd, which was noncytotoxic, radiosensitized HT29 cells up to 48 h after drug removal. During this period, there was an increase in the S phase population, whereas by 72 h after drug removal, the cell cycle distribution resembled that seen under control conditions. dATP pools remained depleted throughout the 72-h period after drug treatment. This study supports the hypothesis that radiosensitization occurs in cells that are replicating DNA in the presence of perturbed dNTP pools. Furthermore, they may be useful in the design of rational clinical trials using dFdCyd as a radiation sensitizer.

Phenotypic correction of dwarfism by constitutive expression of growth hormone.

GH is normally secreted in a pulsatile fashion. When GH is deficient, dwarfism is the result in both rodents and humans. An adenoviral vector containing the rat GH complementary DNA was used to induce constitutive GH expression in hepatocytes of GH-deficient lit/lit mice. Elevated serum GH increased circulating insulin-like growth factor I concentrations, corrected the growth deficiency, and normalized body composition. The results indicate that correction of the dwarf phenotype can be achieved by constitutive expression of GH at an ectopic site by gene transfer.

Radiosensitization of pancreatic cancer cells by 2',2'-difluoro-2'-deoxycytidine.

PURPOSE: We have reported that the deoxycytidine analog 2',2'difluoro-2'-deoxycytidine (dFdCyd) is a potent radiosensitizer of HT29 human colon cancer cells probably through its effects on intracellular deoxyribonucleotide (dNTP) pools. Because dFdCyd has activity against pancreatic cancer in clinical trials, we wished to determine if dFdCyd would radiosensitize human pancreatic cancer cells. METHODS AND MATERIALS: We assessed the effect of dFdCyd on radiation sensitivity of two human pancreatic cancer cell lines, Panc-1 and BxPC-3. To begin to investigate the mechanism of sensitization, we determined the effect of dFdCyd on dNTP pools and cell cycle distribution. RESULTS: We found that dFdCyd produced radiation enhancement ratios of 1.7-1.8 under noncytotoxic conditions in both cell lines. Sensitization was not associated with intracellular levels of 2',2'-difluoro-2'-deoxycytidine triphosphate, the cytotoxic metabolite of dFdCyd, but occurred when dATP pools were depleted below the level of approximately 1 micromolar. Although both cell lines showed substantial cell cycle redistribution after drug treatment, the flow cytogram of the BxPC-3 cells would not, by itself, be anticipated to result in increased radiation sensitivity. CONCLUSIONS: These findings demonstrate that dFdCyd is a potent radiation sensitizer of human pancreatic cancer cells and support the development of a clinical protocol using combined dFdCyd and radiation therapy in the treatment of pancreatic cancer.

Metabolism of 2',2'-difluoro-2'-deoxycytidine and radiation sensitization of human colon carcinoma cells.

Difluorodeoxycytidine (dFdCyd) is a new antimetabolite with clinical activity in patients with solid tumors but not leukemias. We have studied the metabolism, cytotoxicity, and radiosensitizing properties of dFdCyd in HT-29 human colon carcinoma cells. The results demonstrated that dFdCyd rapidly accumulated as the 5'-triphosphate dFdCTP in HT-29 cells, which was eliminated slowly in the absence of dFdCyd with a half-life of > 12 h. Accumulation of dFdCTP was associated with rapid depletion of cellular dATP pools. Exposure to the concentration that reduces cell survival by 50% of 30 nM dFdCyd decreased dATP levels by > 80% within 4 h. dGTP pools were depleted at higher concentrations of dFdCyd, whereas smaller decreases were effected in dTTP and dCTP pools. These results contrast with previous reports in leukemic cells which demonstrated that dFdCyd exposure depleted the endogenous dCTP pool to a greater extent than the dTTP, dATP, or dGTP pools. Based on these data, we suggest that the profound depletion by dFdCyd of dATP and dGTP pools in HT-29 compared to leukemic cells accounts for the superiority of this agent in solid tumors versus leukemias. Additional studies demonstrated that dFdCyd was a potent radiosensitizer in HT-29 cells. Maximal radiosensitization was observed when cells were irradiated immediately following dFdCyd exposure instead of prior to or in the middle of drug treatment. Radiation sensitization was dose and time dependent, with a noncytotoxic exposure to 10 nM dFdCyd for 24 h or 30 nM dFdCyd for 16 h producing a radiation enhancement ratio of approximately 2. Under these conditions, only the cellular dATP pool was depleted. When cells were exposed to higher concentrations of dFdCyd for 4 h, equivalent radiosensitization with a radiation enhancement ratio of 1.4 was obtained using 0.1, 1.0, or 10 microM dFdCyd. Despite a 15-fold increase in dFdCTP and depletion of dGTP and dCTP pools to < 25% of the control value with 10 microM compared to 0.1 microM dFdCyd, no increase in radiosensitization was observed. These results suggest that dATP depletion is an important factor in the radiosensitizing property of this promising new antitumor compound.

Functional characterization of a unique liver gene promoter.

Human phenylalanine hydroxylase (PAH) is specifically expressed in the liver to convert L-phenylalanine to L-tyrosine. Deficiency of the PAH enzyme causes classic phenylketonuria, a common genetic disorder. The human PAH gene has a TATA-less promoter with multiple transcriptional initiation sites. A 9-kilobase DNA fragment 5'-flanking to the human PAH gene is sufficient to confer tissue- and developmental stage-specific expression of a reporter gene in transgenic mice. Deletion studies showed that the -121-base pair proximal promoter still retained a significant level of activity in hepatic cells. At least two protein binding sites, PAH-A and PAH-B, were identified in the proximal region of the human PAH promoter using rat liver nuclear extract. The PAH-A site covers a unique palindromic sequence, and the PAH-B site contains CCCTCCC repeats. Both elements are ubiquitous and essential regulatory elements for transcriptional activity. Nuclear protein factors that bind to the PAH-A and -B sites are detected in different cell types and are distinct from previously characterized transcription factors. No tissue-specific transcription factor binding sites have been detected within the proximal promoter region of the human PAH gene. These results suggest that the PAH gene promoter has a unique organization of regulatory elements for its tissue-specific expression in comparison with other liver gene promoters.

Tissue- and development-specific expression of the human phenylalanine hydroxylase/chloramphenicol acetyltransferase fusion gene in transgenic mice.

Human phenylalanine hydroxylase (PAH) catalyzes the conversion of L-phenylalanine to L-tyrosine. Deficiency of this enzyme results in phenylketonuria, a common genetic disorder of amino acid metabolism that causes severe mental retardation. In primates, PAH is expressed specifically in the liver, while in rodents PAH activity is also present in kidney, although at a much lower level. A 9-kilobase genomic DNA fragment at the 5' end of the hPAH gene (hPAH) was fused to the bacterial chloramphenicol acetyltransferase (CAT) gene. The hPAH/CAT minigene was used to generate multiple transgenic mouse lines. In all expressing lines, CAT activity was detected predominantly in the liver and at much lower levels in the kidney. By immunohistochemical staining, CAT expression was localized to hepatocytes and renal epithelial cells, both of which also express the endogenous mouse PAH enzyme. Furthermore, both the transgene and the endogenous mouse PAH were activated at about the same stage of embryonic development in the mouse liver. These results suggest that the 9-kilobase DNA fragment flanking the 5' end of the human PAH gene contains all the necessary cis-acting elements to direct tissue- and developmental-specific expression in vivo.

Phenylalanine hydroxylase deficiency caused by a single base substitution in an exon of the human phenylalanine hydroxylase gene.

A novel restriction fragment length polymorphism in the phenylalanine hydroxylase (PAH) locus generated by the restriction endonuclease MspI was observed in a German phenylketonuria (PKU) patient. Molecular cloning and DNA sequence analyses revealed that the MspI polymorphism was created by a T to C transition in exon 9 of the human PAH gene, which also resulted in the conversion of a leucine codon to a proline codon. The effect of the amino acid substitution was investigated by creating a corresponding mutation in a full-length human PAH cDNA by site-directed mutagenesis followed by expression analysis in cultured mammalian cells. Results demonstrate that the mutation in the gene causes the synthesis of an unstable protein in the cell corresponding to a CRM- phenotype. Together with the other mutations recently reported in the PAH gene, the data support previous biochemical and clinical observations that PKU is a heterogeneous disorder at the gene level.