Edward R. Seidel

Visfatin expression is elevated in normal human pregnancy

Visfatin is a novel-secreted 52kDa adipokine that appears to mimic the action of insulin, inducing glucose transport into mammalian cells. We examined visfatin expression in a cohort of pregnant women to determine if pregnancy influenced visfatin gene expression, circulating levels of visfatin, or local concentrations of visfatin in either omental fat or placenta. Samples of female omental fat, blood and placenta were collected over a 2-year period and frozen at -80 degrees C until they were employed in a series of various assays. Samples were collected during delivery in pregnant women, at hysterectomy in lean women and at bariatric surgery in obese and obese diabetic women. Visfatin expression and concentrations were measured in four cohorts of women: lean controls, pregnant women at term, obese (BMI>40) and obese diabetic women (BMI>40). Visfatin expression was seven times higher in omental fat of pregnant women than in lean women. Immunohistochemistry (IHC) demonstrated that the visfatin gene transcript was translated to protein. An immunoblot confirmed that visfatin protein was much higher in pregnant women than in obese women. Serum visfatin was 20.8ng/ml (n=7) in lean women as compared to 40.3ng/ml in pregnant women (n=4); thus the increased visfatin mRNA levels in omental fat were not reflected in increased serum visfatin. We measured visfatin mRNA content of human placenta and found that placenta expresses substantial amounts of visfatin. GAP-DH, a housekeeping gene that is highly expressed in most human cells had a threshold value (Ct) of 20.9 versus a Ct of 22.4 for visfatin. Again, IHC confirmed that placental visfatin mRNA was translated into visfatin protein. [(3)H] 2-deoxyglucose transport was measured in partially differentiated 3T3-L1 preadipocytes. At a concentration of 2nM, visfatin and insulin produced nearly identical increases in glucose transport. Taken together, these data suggest there is a selective increase in visfatin gene expression in pregnant women at term. Since visfatin also potently and efficaciously induced glucose transport in a cell culture model, any hypothetical role for visfatin in pregnancy should include the possibility that it may function in regulation of maternal/fetal glucose metabolism or distribution.

Gastrointestinal polyamines and regulation of mucosal growth and function

The polyamines, putrescine, spermidine, and spermine, are a group of ubiquitously distributed organic cations that are intimately involved in proliferation. In eukaryotic cells, the parent polyamine, the diamine putrescine, is synthesized from the amino acid omithine by the enzyme omithine decarboxylase (ODC). Sequential addition of two propylamine groups that are supplied by decarboxylated sadenosylmethionine results in synthesis of the higher polyamines spermidine and spermine (Fig. 1). In contrast, prokaryotes express three decarboxylases whose action yields polyamines. In addition to ODC, arginine decarboxylase also yields putrescine and lysine decarboxylase acts in prokaryotes to synthesize cadaverine from lysine. As mammalian cells have retained only one enzyme, ODC, the presence of cadaverine in mammalian tissues serves as a marker for polyamines derived from prokaryotic metabolism. The pKa’s of the amino groups of polyamines are in the range of 10 to 11 and at the physiological pH of 7.4, polyamines exist as highly charged polyacations. All mammalian cells express the enzyme ODC and are able to synthesize polyamines. Cells of the gastrointestinal epithelium, however, are in the unusual position of having both endogenously synthesized polyamines available for metabolism and a large supply of exogenous polyamines available from the gut lumen. The functions of endogenously synthesized polyamines by eukaryotic cells and the relationship of polyamines to cellular proliferation has been the subject of several excellent recent reviews’,‘33 and will not be discussed further. This article, instead, will concentrate on a review of polyamines in the lumen of the gastrointestinal tract and will be divided into sections

Obestatin and ghrelin in obese and in pregnant women.

We identified, through qPCR, receptor mRNA for a number of gut peptides in female human omental fat: the incretins, GIP and GLP-1, the orexigenic peptides PYY-Y1 and -Y2 and ghrelin, and the anorexigenic peptide obestatin. Four cohorts of women were examined: lean controls (BMI<23), obese (BMI>41), obese diabetic and term pregnant women. Human fat expressed receptor mRNAs for all six peptides. Pregnant women expressed roughly three times as much orphan GPR-39 receptor, a proposed obestatin receptor, than other women and less than half as much of the ghrelin receptor (GHSR-1a). An immunoblot probed with a GPR-39 selective antibody yielded a single band corresponding to the correct molecular weight (52 kDa) for the proposed obestatin receptor. Fluorescent immunohistochemistry of human fat employing the same antibody indicated the receptor protein was localized to the adipocyte cell membrane. The concentration of obestatin circulating in blood was measured in the same cohort of women and was significantly lower in obese and obese diabetic women compared to control.

Vascular endothelial cell proliferation: regulation of cellular polyamines.

OBJECTIVE The aims were to investigate the requirement for and regulation of cellular polyamines during vascular endothelial cell proliferation. METHODS Proliferation of cultured bovine pulmonary artery endothelial cells was determined after cellular polyamine depletion. In addition, polyamine synthesis and uptake mechanisms were examined in the presence and absence of trophic stimuli and density dependent inhibition of cell proliferation. RESULTS Serum stimulated, subconfluent cells ceased cell division following the inhibition of ornithine decarboxylase (ODC), a rate limiting enzyme for polyamine biosynthesis. The addition of 10 microM putrescine, the product of the enzyme reaction catalysed by ODC, completely reversed the inhibition of cell growth. Serum deprivation reduced cytosolic ODC activity to near non-detectable levels. Readdition of 10% fetal bovine serum (FBS) resulted in transient increases in ODC activity which preceded DNA synthesis and mitosis. Basic fibroblast growth factor also stimulated ODC activity in a dose dependent manner with levels approximating the maximum obtainable with FBS. In contrast, platelet derived growth factor and epidermal growth factor did not stimulate ODC activity. Finally, mitogenic stimuli did not induce ODC activity in density arrested cells. The uptake of radiolabelled putrescine from the cell medium was time dependent and saturable. Kinetic studies from dividing cells revealed a single transport component for putrescine uptake [maximum rate of uptake (Vmax) = 11.2(SEM 2.0) pmol.10(5) cells-1.h-1; Michaelis constant (Km) = 1.1(0.3) microM]. Putrescine uptake by density arrested cells was characterised by a 57% decrease in Vmax with no change in Km. Readdition of FBS to serum deprived subconfluent cells, in the presence of ODC inhibitors, resulted in a rapid increase in the rate of putrescine uptake with Vmax increasing by 533% over FBS alone by 48 h. DISCUSSION These data suggest that polyamines are essential for endothelial cell proliferation and that synthesis and uptake mechanisms are regulated according to cell growth.

Insulin-like growth factor I in human gastrointestinal exocrine secretions.

Insulin-like growth factor I (IGF-I) is the mediator of growth hormone dependent growth. The peptide has been identified by radioimmunoassay in a number of human exocrine secretions of the gastrointestinal tract including (nM): saliva 0.9, gastric juice 3.5, jejunal chyme 24.6, pancreatic juice 3.6, and bile 0.9. The identification of IGF-I in pancreatic juice was confirmed by HPLC. The intravenous injection of 1 unit/kg secretin increased pancreatic juice IGF-I content from a basal level of roughly 4 nM to nearly 20 nM. Conversely, the IGF-I content of bile was unaffected by secretin. Radioligand blot analysis of samples of gastric juice, jejunal chyme and pancreatic juice demonstrated that these fluids contained no IGF binding proteins. Thus, unlike IGF-I in serum, IGF-I secreted into the gastrointestinal lumen is not bound to insulin-like growth factor I binding proteins. Since the growth factor is not protein bound, its concentration in the gut lumen may be high enough to exert biological activity.

Estrogens induce visfatin expression in 3T3-L1 cells

Visfatin is a 56 kDa protein that is overexpressed in pregnant women. Like insulin, 2 nM visfatin induced GLUT 4 translocation from the cytosolic fraction to the membrane in 3T3-L1 cells. We have previously reported that visfatin induces glucose uptake into 3T3-L1 cells. These two actions define visfatin as an insulinomimetic. Three estrogens are elevated in pregnancy. Estradiol, the predominant estrogen, estriol, produced by the fetal liver and the pro-estrogen progesterone are all higher during pregnancy than in nonparous women. 3T3-L1 cells were treated with 150 ng/ml estriol, 16 ng/ml estradiol or 190 ng/ml progesterone to reflect the circulating concentrations of these steroids during pregnancy. Estriol treatment produced a 2.5-fold increase in visfatin gene expression. Estradiol and progesterone had small but insignificant effects on visfatin gene expression. In a second experiment, cells were treated with a combination of all three steroids together at the same concentrations listed above. The combination treatment produced a 13-fold increase in visfatin gene expression. These data suggest that the estriol, estradiol and progesterone exert a synergistic effect on visfatin gene expression. Taken together these data suggest that visfatin may play a physiological role during pregnancy. Since visfatin potently and efficaciously induced GLUT 4 translocation in a cell culture model, any hypothetical role for visfatin in pregnancy should include the possibility that it may play a role in maternal/fetal glucose metabolism or distribution. Two possibilities present: either maternal visfatin is overexpressed as a protective response in the pregnant female to compensate for the insulin resistance that often accompanies pregnancy or the excess visfatin is a compensatory response to ensure adequate glucose delivery to the growing fetus.

Insulin-like growth factor I in suckling rat gastric contents

The small intestinal mucosa of the neonatal rat expresses primarily lactase activity until just prior to weaning when lactase falls to low levels and a full complement of adult digestive enzymes appears. Insulin-like growth factor 1 (IGF-I) is a normal component of maternal milk of humans and experimental animals. Experiments were performed to examine the concentrations of IGF-I in dam milk and the gastric content of suckling pups. Lactase activity in 1-day-old neonates was 0.66 µmol glucose formed/mg protein/hr (unit) and fell progressively until day 25, whereas sucrase activity at day 1 postpartum was 0.07 units and rose progressively to 0.21 units at day 25. The IGF-I content of dam milk was measured at 1, 5, 10, 15, 18, and 20 days postpartum by radioreceptor assay (RRA). Milk contained 1.02 pmol IGF-I/ml milk at one day postpartum, peaked at day 18 with 5.08 pmol IGF-I/ml, and fell to 2.31 pmol/ml at day 20. By day 25, dams were dry. The IGF-I content of the neonate gastric lumen was also measured by RRA. At day 1 the gastric lumen contained 2.63 pmol IGF-I/ml of luminal contents, fell to 1.06 pmol IGF-I/ml at day 5, and then rose again to peak at 3.37 pmol/ml at day 15 just prior to weaning. Two days after weaning, the level of luminal IGF-I had fallen to 1.15 pmol/ml. These data demonstrate the concentration of IGF-I in maternal milk is reflected in the concentration of the peptide in gastric contents of suckling pups and that the concentration in the gastric lumen may be high enough to affect epithelial cell proliferation and differentiation.

Polyamines are absorbed through a y+ amino acid carrier in rat intestinal epithelial cells

Summary.Due to the similarity in transport characteristics of polyamines and the y+ basic amino acid system, we hypothesized that both substrates could be moving through a common carrier site. Competitive and cross inhibition experiments in intestinal epithelial cells revealed the possibility of a common transport site. N-ethylmalemide (NEM) inhibited both lysine and putrescine transport, confirming that both were carried by a y+ transporter. Overexpressing the y+ transporter CAT-1 in a polyamine transport-deficient cell line, CHO-MG, did not reconstitute polyamine-transport. Thus, polyamines are not traveling through CAT-1. To determine if lysine is carried by a polyamine transport site, an antizyme-overexpressing cell line was used. Antizyme overexpression decreased polyamine uptake by 50%; in contrast, lysine transport was unaffected. Therefore, lysine is not traveling through a polyamine transport site. It appears that polyamines and lysine are likely traveling through a common unknown y+ transport site.

Selective sites for polyamine binding to rabbit intestinal brush-border membranes

The intestinal polyamine transporters have not yet been identified. Our aim was to characterize specific polyamine binding sites in rabbit intestinal brush-border membranes (IBBM) as a starting step for identification of polyamine transporters. This was investigated at 4 degrees and at low membrane concentration. Saturation isotherms for [3H]putrescine (PUT) binding indicated a single population of sites (puT) with a dissociation equilibrium constant Kd of 3.8 microM and a density of sites Bmax of 58 pmol/mg of protein. [3H]spermidine (SPD) binding also involved only one class of sites (spD), albeit with a lower affinity (Kd = 106 microM) and higher abundance (Bmax = 1240 pmol/mg of protein) than puT. On the contrary, [14C]spermine (SPM) bound two classes of sites (spM1 and spM2) differing in their affinity (Kd = 2.5 and 31.4 microM) and abundance (Bmax = 467 and 1617 pmol/mg of protein, respectively). Membrane association of SPM at 4 degrees was much faster than that of SPD and PUT, both of which proceeded at a similar rate. In contrast to PUT and SPD dissociation, SPM dissociation at 23 degrees did not follow a first-order reaction. Specifically bound [3H]PUT, unlike [3H]SPD and [14C]SPM, dissociated at 23 degrees independently of the addition of nonradioactive polyamine. Methylglyoxal-bis-(guanylhydrazone) was an extremely potent inhibitor of PUT binding (Ki = 3.2 +/- 1.5 nM), but as with PUT and cadaverine (CAD), it did not alter [3H]SPD and [14C]SPM binding substantially. The intestinal brush-border membrane may contain at least three sites specific for polyamine binding and exhibiting different ligand selectivity. Site puT might be associated with the transport system already described for intestinal uptake of PUT.

Peroxynitrite inhibits the activity of ornithine decarboxylase

Polyamines are required during cell proliferation, whereas NO has anti-proliferative properties. Ornithine decarboxylase (ODC) is a critical enzyme for the synthesis of polyamines. We tested the hypothesis that the modification of ODC by peroxynitrite (OONO-), a short-lived free radical formed from NO and superoxide produces a fall in ODC activity, and therefore polyamine synthesis and cell proliferation. The treatment of a rat recombinant ODC (rODC) with OONO- resulted in a dose-dependent inhibition of rODC activity with an IC50 of approximately 100 microM. A Western blot employing a specific antibody to nitrotyrosine revealed a dose-dependent nitration of rODC tyrosine residues. When intact IEC-6 cells were treated with ONOO-, ODC activity decreased by 49%. These data suggest a correlation between ODC activity and nitration, and a possible mechanism by which NO synthesis may modulate polyamine synthesis.