[11C]WAY 100635: A radioligand for imaging 5-HT1A receptors with positron emission tomography

The potent and selective 5-HT_1A antagonist WAY 100635 (N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexanecarboxamide) was radiolabeled with ¹¹C in high specific activity, and the in vivo properties of this radioligand were assessed in the brains of rats and monkeys. Following i.v. tail vein injection in rats, [¹¹C]WAY 100635 rapidly penetrated into brain tissue and was retained over a 30–90 min time period in a manner consistent with the known distribution of 5-HT_1A receptors. Pretreatment of rats with the selective 5-HT_1A agonist (±)-8-OH-DPAT effectively blocked the retention of radioactivity in brain regions known to contain high densities of 5-HT_1A receptors. The hippocampus-to-cerebellum radioactivity concentration ratio reached a maximum of 16:1 at 60 min post injection. Following i.v. injection of [¹¹C]WAY 100635 in rhesus monkeys, the concentrations of radioactivity in brain regions were consistent with the reported distribution of 5-HT_1A receptors in primates, and the frontal cortex-to-cerebellum ratio reached 5.5:1 at 80 min post injection. Pretreatment of the monkeys with (±)-8-OH-DPAT reduced this ratio to 1.4:1, and injection of (±)-8-OH-DPAT 20 min after the injection of [¹¹C]WAY 100635 significantly displaced frontal cortex binding. The in vivo properties of [¹¹C]WAY 100635 in rats and monkeys strongly support the future utility of this radioligand for imaging 5-HT_1A receptors using positron emission tomography (PET).     

Rate of incorporation of [3H]thymidine in various tissues of the mouse. A basis for the evaluation of genotoxic effects of chemicals

[3H]Thymidine has been extensively used as a selective precursor to DNA in studies on the kinetics of cell proliferation. We have become interested in measuring early inhibition of the DNA synthesis in various organs of intact animals for detecting genotoxic properties of chemicals. Such experiments should, for convenience and to achieve a large capacity, be performed in the simplest way possible. The present paper deals with some practical aspects on the use of [3H]thymidine in vivo. [6-3H]Thymidine was injected intraperitoneally in mice and the uptake of radioactivity was evaluated by using whole-body autoradiography and liquid scintillation spectrometry. Autoradiograms of sections washed with trichloroacetic acid and methanol were compared with those subjected only to freeze-drying. Liquid scintillation counting was performed of total, non-volatile, acid-insoluble and DNA-associated radioactivities. A rapid increase of the [3H]thymidine incorporation was seen during the first hour after the injection. Further prolongation of the survival time did not result in any significant increase of the incorporated radioactivity. Moreover, there were only slight differences between the autoradiograms from extracted and non-extracted sections. Radioactivities associated with DNA closely correlated to those representing acid-insoluble material, indicating that acid-insoluble radioactivity provides a good estimate of the [3H]thymidine incorporation into DNA.     

4-[18F]Fluoro-L-m-Tyrosine: An L-3,4-Dihydroxyphenylalanine Analog for Probing Presynaptic Dopaminergic Function with Positron Emission Tomography

4-[18F]Fluoro-L-m-tyrosine (FMT), a biochemical probe of striatal dopaminergic function, has been synthesized as an L-3,4-dihydroxyphenylalanine analog for positron emission tomography. Biochemical characterization of this compound in the rat 30 min after intrajugular administration indicated that in the brain, selective decarboxylation occurred in the striatum, with the formation of 4-fluoro-3-hydroxyphenylethylamine and its metabolites. Positron emission tomography analysis of brain tissue in monkeys (Macaca nemestrina) after intravenous injection of FMT revealed a true time-dependent, specific accumulation of radioactivity in striatum, with a striatum/cerebellum (nonspecific) ratio of 4 at 180 min. Peripheral metabolism accounted for less than 40% of the total radioactivity in arterial blood samples after 120 min. The amino acid remained as the major component throughout the period of investigation (n = 3; 5 min, 95%; 10 min, 85%; 30 min, 67%; 60 min, 62%; 120 min, 60%), with a plasma clearance t 1/2 of 112 min. 3-O-Methylated metabolites were not observed. The substrate specificity of FMT, coupled with its limited in vivo peripheral metabolism, makes it a useful, new biochemical probe for in vivo, noninvasive evaluation of central dopaminergic mechanisms.     

Distribution of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in experimental animals studied by postiron emission tomography and whole body autoradiography

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a selective potent neurotoxin which has induced a syndrome similar to parkinsonism both in man and in monkeys. At autopsy degeneration of pigmented nerve cells in the pars compacta of the substantia nigra has been confirmed. The regional distribution of intravenously administered 1-(11C-methyl)-4-phenyl-1,2,3,6-tetrahydropyridine (11C-MPTP) in the brain of Rhesus monkeys was studied by positron emission tomography and the whole body distribution in mice was documented by autoradiography and by impulse counting of selected tissues. A very rapid and high uptake of 11C-MPTP derived radioactivity was seen in areas corresponding to striatum and midbrain, including the substantia nigra area. No elimination from these regions was seen during the study period of 2 h. The uptake was in the order of 7-8 times the homogenous distribution of the radioactivity in the monkey. The uptake was generally high also in other regions of the brain, but there some elimination could be distinguished. Pretreatment of the monkey with spiperone, a selective dopamine receptor antagonist, did not alter uptake nor the kinetics of the 11C-MPTP derived radioactivity. Thus 11C-MPTP does not have a high affinity for postsynaptic dopamine receptors. A remarkably high uptake of 11C-MPTP derived radioactivity was seen in the eye of the monkey. The selective uptake of radioactivity in the eye was also confirmed in pigmented but not in albino mice. The melanin affinity of MPTP may cause high intracellular concentrations of the compound or its metabolites in the melanin containing nerve cells in substantia nigra, which may explain the serious vulnerability of these neurons to MPTP.     

Kinetics of four 11C-labelled enkephalin peptides in the brain, pituitary and plasma of rhesus monkeys

The kinetics of four 11C-labelled enkephalin peptides: Tyr-Gly-Gly-Phe-Met (Met-enkephalin), Tyr-D-Met-Gly-Phe-Pro-NH2 [D-Met2,Pro5)-enkephalinamide), Tyr-D-Ala-Gly-Phe-Met-NH2 (DALA) and Tyr-D-Ala-D-Ala-Phe-Met-NH2 (TAAFM) all labelled at the methyl group of methionine was studied in the Rhesus monkey. After intravenous administration, the regional kinetics in the head, lungs, liver and kidneys were followed by means of positron emission tomography (PET). The total radioactivity in blood and urine was measured and the composition of 11C-labelled peptide fragments in plasma in vivo and in vitro was analysed by liquid chromatography. With PET, an increased radioactivity was observed in the brain and pituitary over the 60-90 min investigation period after i.v. injection of the peptides. The highest radioactivities were noted for Met-enkephalin, followed by DALA and D-Met2, Pro5-enkephalinamide, while very low radioactivities were found for TAAFM. The uptake of Met-enkephalin- and DALA-derived radioactivity was of the same order as has previously been shown for morphine in the brain and considerably higher than that of D-Met2,Pro5-enkephalinamide and TAAFM, respectively. A large fraction of the brain radioactivity derived from Met-enkephalin and DALA probably emanated from [11C]methionine as indicated by plasma and urine analysis. Met-Enkephalin was rapidly eliminated from plasma in vitro with an half-life of less than two minutes, whereas DALA was stable suggesting clearance by other tissues than plasma. In conclusion, both Met-enkephalin and DALA, were rapidly hydrolyzed in vivo to [11C]methionine. [11C]Methionine was probably taken up in the brain, as the radioactivity increased with time in different brain regions as measured with PET.D-Met2,Pro5-Enkephalinamide and TAAFM were virtually stable in vivo and at least part of the radioactivity observed in the brain may have represented the intact peptide.     

Rate of Incorporation of R3hlthymidine In Various Tissues of the Mouse A basis for the evaluation of genotoxic effects of chemicals

Abstract. [³H]Thymidine has been extensively used as a selective precursor to DNA in studies on the kinetics of cell proliferation. We have become interested in measuring early inhibition of the DNA synthesis in various organs of intact animals for detecting genotoxic properties of chemicals. Such experiments should, for convenience and to achieve a large capacity, be performed in the simplest way possible.
The present paper deals with some practical aspects on the use of [³H]thymidine in vivo. [6-³H]Thymidine was injected intraperitoneally in mice and the uptake of radioactivity was evaluated by using whole-body autoradiography and liquid scintillation spectrometry. Autoradiograms of sections washed with trichloroacetic acid and methanol Were compared with those subjected only to freeze-drying. Liquid scintillation counting was performed of total, non-volatile, acid-insoluble and dNA-associated radioactivities. A rapid increase of the [³H]thymidine incorporation was seen during the first hour after the injection. Further prolongation of the survival time did not result in any significant increase of the incorporated radioactivity. Moreover, there were only slight differences between the autoradiograms from extracted and non-extracted sections. Radioactivities asśociated with DNA closely eorrelated to those representing acid-insoluble material, indicating that acid-insoluble radioactivity provides a good estimate of the [³H]thymidine incorporation into DNA.     

Mapping of Histamine H1 Receptors in the Human Brain Using [11C]Pyrilamine and Positron Emission Tomography

We have studied the characteristics of carbon-11 labeled pyrilamine as a radioligand for investigating histamine H1 receptors in human brain with positron emission tomography (PET). [11C]Pyrilamine is distributed evenly in proportion to cerebral blood flow at initial PET images. Later (after 45-60 min), 11C radioactivity was observed at high concentrations in the frontal and temporal cortex, hippocampus, and thalamus, and at low concentrations in the cerebellum and pons. The regional distribution of the carbon-11 labeled compound in the brain corresponded well with that of the histamine H1 receptors determined in vitro in autopsied materials. In six controls, the frontal and temporal cortices/cerebellum ratio increased during the first 60 min to reach a value of 1.22 +/- 0.071. Intravenous administration of d-chlorpheniramine (5 mg) completely abolished the specific binding in vivo in the frontal cortex and temporal cortex (cortex/cerebellum ratio, 0.955 +/- 0.015). The availability of this method for measuring histamine H1 receptors in vivo in humans will facilitate studies on neurological and psychiatric disorders in which histamine H1 receptors are thought to be abnormal.     

Comparison of Three 18F-Labeled Butyrophenone Neuroleptic Drugs in the Baboon Using Positron Emission Tomography

The butyrophenone neuroleptics spiroperidol, benperidol, and haloperidol were radiolabeled with fluorine-18 and studied in baboon brain using positron emission transaxial tomography (PETT). Pretreatment of the baboon with a high pharmacological dose of (+)-butaclamol reduced the specifically bound component of radioactivity distribution in the striatum to approximately the radioactivity distribution found in the cerebellum. Comparative studies of brain distribution kinetics over a 4-h period indicated that either [18F]spiroperidol or [18F]benperidol may be suitable for specific labeling of neuroleptic receptors. In an 8-h study with [18F]spiroperidol, striatal radioactivity did not decline, suggesting that spiroperidol either has a very slow dissociation rate or that it binds irreversibly to these receptors in vivo. [18F]Haloperidol may not be suitable for in vivo PETT studies, because of a relatively high component of nonspecific distribution and a faster dissociation from the receptor. Analysis of 18F in plasma after injection of [18F]spiroperidol indicated rapid metabolism to polar and acidic metabolites, with only 40% of the total radioactivity being present as unchanged drug after 30 min. Analysis of the metabolic stability of the radioactively labeled compound in rat striatum indicated that greater than 95% of [18F]spiroperidol remains unchanged after 4 h.     

Plasma drug levels compared with DNA incorporation of 3'-azido-3'-deoxythymidine (AZT) in adult cynomolgus (Macaca fascicularis) monkeys

Zidovudine (3'-azido-3'-deoxythymidine, AZT), widely used for the therapy of the Human Immunodeficiency Virus-1 (HIV-1), is a nucleoside analog of thymidine that becomes phosphorylated and incorporated into nuclear and mitochondrial DNA. Levels of AZT incorporation into DNA of humans, monkeys, and mice are highly variable and suggest interindividual variability in phosphorylation pathways. In addition, studies in rhesus monkeys (1) have shown a lack of correlation between levels of unbound AZT in plasma and tissue AZT-DNA. However, the correlation between plasma AZT and tissue AZT-DNA has not been previously examined in the same primate. Here we examine the relationship between AZT-DNA incorporation in leukocytes and multiple organs, and levels of the drug circulating in plasma of adult female cynomolgus (Macaca fascicularis) monkeys. Three monkeys were dosed with 40.0 mg of AZT/day for 30 days by naso-gastric intubation. The average daily dose of 9.9 mg of AZT/kg/body wt was similar to the approximately 8.6 mg of AZT/kg/body wt (600 mg/day) given to adult HIV-1-infected patients. In all three monkeys, at the time of sampling, values for AZT concentrations in plasma were similar and values for AZT incorporation into leukocyte DNA (86.1, 100.0, and 114.1 molecules of AZT/10(6) nucleotides) were also similar. AZT-DNA incorporation was detected in liver, uterus, spleen, and kidney from the three AZT-exposed animals, with values for positive samples ranging from 5.8 to 97.4 molecules of AZT/10(6) nucleotides. In brain cortex and lung DNA from AZT-exposed animals, AZT incorporation was undetectable. The data suggest that organ-specific differences in AZT uptake and/or metabolism may contribute to AZT phosphorylation and subsequent drug incorporation into DNA. In addition, AZT-DNA levels in monkey organs were similar to or lower than values observed in peripheral leukocytes of adult AIDS patients.     

Tissue distribution of humanized anti-human Fas monoclonal antibody (R-125224) based on fas antigen-antibody reaction in collagen-induced arthritis monkeys

R-125224 is a novel humanized anti-human Fas monoclonal antibody prepared from HFE7A, which is a monoclonal mouse IgG anti-Fas antibody, by grafting the mouse complementarity-determining regions to human IgG, presently being developed as a drug for treatment of rheumatoid arthritis. In the present study, we investigated the tissue distribution of radioactivity in cynomolgus monkeys with collagen-induced arthritis at the arm joint (CIA monkeys) after intravenous administration of (125)I-labeled R-125224 ((125)I-R-125224). At 168 h after administration, we observed a high radioactivity in the bone marrow, thymus, lungs, liver, adrenals, spleen, ovaries, axillary lymph node and mesenteric lymph node compared to the radioactivity in the plasma. These tissues and organs in human are reported to express Fas antigen, strongly suggesting a specific binding of (125)I-R-125224 to Fas antigen in cynomolgus monkeys. Semi-micro autoradioluminograms of arm joint showed that radioactivity is detected in pharmacological site, such as the bone marrow and articular cavity at 168 h. The kinetics in binding of R-125224 to activated monkey lymphocytes and hepatocytes was also investigated. K(d) values of activated lymphocytes and hepatocytes were 1.51+/-0.08 and 0.60+/-0.11 nM, respectively, which were similar to those values in human lymphocytes and hepatocytes, demonstrating that R-125224 cross-reacts with the monkey Fas antigen.     

Synthesis and Characterization of Binding of 5-[76Br] Bromo-3-[[2(S)-Azetidinyl]methoxy]pyridine, a Novel Nicotinic Acetylcholine Receptor Ligand, in Rat Brain

5-[76Br]Bromo-3-[[2(S)-azetidinyl]methoxy]pyridine ([76Br]BAP), a novel nicotinic acetylcholine receptor ligand, was synthesized using [76Br]bromide in an oxidative bromodestannylation of the corresponding trimethylstannyl compound. The radiochemical yield was 25%, and the specific radioactivity was on the order of 1 Ci/micromol. The binding properties of [76Br]BAP were characterized in vitro and in vivo in rat brain, and positron emission tomography (PET) experiments were performed in two rhesus monkeys. In association experiments on membranes of the cortex and thalamus, >90% of maximal specific [76Br]BAP binding was obtained after 60 min. The dissociation half-life of [76Br]BAP was 51 +/- 6 min in cortical membranes and 56 +/- 3 min in thalamic membranes. Saturation experiments with [76Br]BAP revealed one population of binding sites with dissociation constant (K(D)) values of 36 +/- 9 and 30 +/- 9 pM in membranes of cortex and thalamus, respectively. The maximal binding site density (Bmax) values were 90 +/- 17 and 207 +/- 33 fmol/mg in membranes of cortex and thalamus, respectively. Scatchard plots were nonlinear, and the Hill coefficients were <1, suggesting the presence of a lower-affinity binding site. In vitro autoradiography studies showed that binding of [76Br]BAP was high in the thalamus and presubiculum, moderate in the cortex and striatum, and low in the cerebellum and hippocampus. A similar pattern of [76Br]BAP accumulation was observed by ex vivo autoradiography. In vivo, binding of [76Br]BAP in whole rat brain was blocked by preinjection of (S)(-)-nicotine (0.3 mg/kg) by 27, 52, 68, and 91% at survival times of 10, 25, 40, 120, and 300 min, respectively. In a preliminary PET study in rhesus monkeys, the highest [76Br]BAP uptake was found in the thalamus, and radioactivity was displaceable by approximately 60% with cytisine and by 50% with (S)(-)-nicotine. The data of this study indicate that [76Br]BAP is a promising radioligand for the characterization of nicotinic acetylcholine receptors in vivo.     

Uptake and translocation of phytochemical 2-benzoxazolinone (BOA) in radish seeds and seedlings

The molecular aspects of phytochemical interactions between plants, especially the process of phytochemical translocation by the target plant, remain challenging for those studying allelopathy. 2-Benzoxazolinone (BOA) is a natural chemical produced by rye (Secale cereale) and is known to have phytotoxic effects on weed seeds and seedlings. The translocation of BOA into target plants has been poorly investigated. Therefore, the total absorption of [ring U 14C] BOA was estimated by oxidizing whole seedlings of Raphanus sativus cv. for 8 days and quantifying the radioactivity. Non-radiolabelled BOA in seedlings was also estimated by HPLC. BOA applied at 10(-3) M was readily taken up by germinated radish at a rate of 1556 nmol g(-1) FW. At these same concentrations, BOA reduced radish germination by 50% and caused a delay in radicle elongation. Exogenous BOA was responsible for the observed germination inhibition. At a concentration of 10(-5) M, BOA was taken up by germinated seeds (31 nmol g(-1) FW), but this quantity did not affect radish germination. Labelled BOA was not mineralized in the culture medium during seedling growth as no 14CO2 was recovered. Both 10(-3) and 10(-5) M BOA were translocated into radish organs, mainly into roots and cotyledons. These organs were then identified as potential physiological target sites. Cotyledons remained the target sink (44% of the total radioactivity). The kinetics of BOA uptake at 10(-3) and 10(-5) M in radish seedlings was identical: BOA accumulation was proportional to its initial concentration. A comparison between radioactivity and HPLC quantification for 10(-3) M BOA indicated that BOA (along with some metabolites) could effectively be recovered in radish organs using chromatography.     

Routes of administration and uptake of tritiated nucleotides

Summary The uptake, incorporation and distribution of tritiated thymidine and uridine into visceral organs of immature female and male rats was studied. Radioautographs of various organs were prepared 5, 15 and 30 min after intraperitoneal administration. The distribution of the radioactivity was compared with sections obtained 30 min after intravenous injection of the same amount of nucleotides.The intraperitoneal injection was performed by directing the needle toward the right side of the abdominal cavity. Visceral organs from the injected side contained consistently more silver grains than those from the opposite side. Sections of the abdominal organs showed a decreasing concentration gradient of reduced silver grains extending from the serosal part of the organ to the deeper layers. In addition, initial labeling was observed in sectors of organs which protruded into the peritoneal cavity. In contrast, following intravenous administration of nucleotides a concentration gradient was not observed in any of the visceral organs.The results of the present study suggest that nucleotides administered intraperitoneally may penetrate the abdominal organs directly via the serosal surface without prior entry into the general circulation.     

99mTc标记丝状噬菌体肽库及其在正常小鼠体内的分布

利用噬菌体展示技术已选出了多条与靶结合的肽.然而,即使是体内直接筛选得到的,肽与肿瘤或靶器官的体内结合并不理想.为了更好地理解噬菌体在体内的循环,通过MAG399mTc标记噬菌体肽库,研究了肽库在体内分布.体内分布实验结果显示,99mTc-噬菌体主要分布在肝和脾中,而心脏、肌肉、脑和胰腺这些器官或组织中的分布非常低.99mTc-噬菌体在胃、肠和骨中的累积,随着时间延长在不断升高,其他器官中的吸收则在不断降低.从5min到30min,99mTc-噬菌体在血中清除迅速.当噬菌体在体内循环足够长的时间后,一些噬菌体颗粒可以穿透血管进入并内化在器官或组织中.总之,为了筛选具有高特异性和亲和性的肽,应该根据靶器官和筛选部位的不同,在筛选前确定合适的噬菌体在体内的循环时间.     

Synthesis and in vitro/in vivo evaluation of novel 99mTc(CO)3-folates

Novel organometallic 99mTc(I)-folate derivatives have been synthesized and evaluated in vitro and in vivo in order to assess the influence of the overall charge of the radioconjugates and the spacer entity on the affinity and pharmacokinetic profile. Folic acid has been functionalized at the gamma-carboxylate group of the glutamate moiety with (i) a hydrophilic diethoxyethyl spacer bearing a picolylamine monoacetic acid chelate, (ii) a hexyl spacer bearing an iminodiacetic acid chelate, and (iii) a hexyl spacer with a bis(pyridylmethyl)amine chelating system. Coordination of the 99mTc(CO)3-core resulted in neutral complex 21, anionic complex 22, and cationic complex 23 in excellent yields (>90%) at ligand concentrations of 10(-4) M. Complexes 21-23 were HPLC purified for in vitro and in vivo experiments. In the case of 23, separation from the unlabeled folate analogue was incomplete, leading to low specific activity and, hence, significantly inferior in vivo uptake in folate-receptor-positive (FR-positive) organs and tissues (tumors and kidneys). Time dependent in vivo studies were performed in female, athymic nude mice bearing subcutaneous FR-positive human KB cell xenografts at 1, 4, and 24 h post injection (p.i.) of the radiotracers. Tumor uptake ranged between 1.9-2.7% ID/g, 4 h p.i. and 1.6-2.2% ID/g, 24 h p.i. for 21 and 22, and 0.9% ID/g, 4 h p.i. and 1.1% ID/g, 24 h p.i. for 23. Blood clearance was fast for all derivatives (< or =0.2% ID/g 1 h p.i.). Significant fractions of radioactivity were found in nontargeted and FR-negative organs and tissues (particularly in the liver and the intestines/intestinal contents) at early time points p.i. Coadministration of folic acid reduced radioactivity in FR-positive tissues and organs to background levels. In conclusion, overall charge and the nature of the spacer entity seemed to have a relatively minor influence on receptor affinity and the in vivo pharmacokinetic profile of the tested radiofolates.     

Distribution, metabolism and excretion of [1-14C]dolichol injected intravenously into rats.

[1-14C]Dolichol mixed in vitro with rat serum and injected intravenously into rats was rapidly cleared from the circulation in a manner consistent with a two-compartment model. About 80% of the radioactivity recovered from animals killed after 1 day was in the liver, with smaller amounts being found in lung, carcass (internal organs removed), gastrointestinal tract and contents, and spleen. The kidneys, testes and heart contained little radioactivity, and the brain did not appear to take up any [1-14C]dolichol. The half-life for the turnover of radioactivity from [1-14C]dolichol in tissues varied considerably, being 2 days for the lung, 17 for liver and about 50 days for the carcass. After 1 day, and also after 4 and 21 days, most of the radioactivity in all tissues was as [1-14C]dolichol and as [1-14C]dolichyl fatty acyl ester, although a small amount of incorporation of [1-14C]dolichol radioactivity into phospholipids was also observed. Faeces collected over the first 4 days after injection contained 13% of the [1-14C]dolichol dose, but urine and expired air contained only small amounts of radioactivity. Radioactivity in faeces was nearly all as unchanged [1-14C]dolichol and as [1-14C]dolichyl fatty acyl ester. The [1-14C]dolichol remaining in liver after 21 days appeared to be in a pool (possibly lysosomes) where most of it was not subject to excretion.     

Insulin Levels,Physical Activity,and Urinary Catecholamine Excretion of Obese and Non-Obese Rhesus Monkeys

The hypothesis that spontaneous obesity in rhesus monkeys is associated with abnormalities in energy expenditure was tested. Obese (n=7) and non-obese (n=5) monkeys were described in terms of body size and composition, food intake, and physical activity. Additionally, the relationships among fasting and stimulated insulin levels in serum, C-peptide levels in serum and urine, and urinary catecholamines were examined. Obese animals had primarily abdominal deposition of excess body fat, as indicated by markedly elevated abdominal circumferences and skin-fold thicknesses. Food intake did not differ between groups. Physical activity was much lower in the obese group. Obese monkeys had markedly higher serum insulin and C-peptide levels in the fasted state and in response to an intravenous glucose challenge. Urinary excretion of C-peptide and catecholamines was measured during successive 2-day periods of ad libitum feeding, food deprivation, and refeeding in order to examine potential differences between groups in sympathoadrenal activity and their relationship to insulin secretion. C-peptide excretion was greater for obese and decreased for both groups during food deprivation. Urinary dopamine (DA), norepinephrine (NE), and epinephrine (E) levels were significantly greater for obese animals in all conditions. DA excretion was lowest during deprivation and E excretion was lowest during refeeding, whereas NE excretion was relatively unaffected by feeding condition. The overall patterns of C-peptide and catecholamine excretion were qualitatively similar for both groups, and there were no reliable differences between obese and non-obese in their responses to the feeding manipulation. The results suggest that hyperinsulinemia associated with obesity in rhesus monkeys is linked to increased catecholamine secretion and a resistance to cate-cholaminergic action.     

Evidence for heterogeneity of low-density lipoprotein metabolism in the cynomolgus monkey

Preliminary studies were performed to establish whether there was kinetic heterogeneity in the metabolism of subclasses of low-density lipoproteins (LDL) in the cynomolgus monkey. Previous studies of the effects of inhibition of hepatic triglyceride lipase in this species had shown an increase in the mass of lighter LDL (Sf greater than 9) and a decrease in the mass of denser LDL. LDL (1.019 less than d less than 1.063) were subdivided into two subfractions LDL1 (1.019 less than d less than 1.035) and LDL2 (1.035 less than d less than 1.063) by ultracentrifugation. The lipoproteins in these two fractions could be shown to have different flotation by analytic and isopycnic ultracentrifugation. When tracer amounts of homologous 125I-labeled very-low-density lipoproteins (VLDL) were injected into chow-fed cynomolgus monkeys, apoB radioactivity appeared in LDL1 prior to its appearance in LDL2. [125I]LDL1 injected into the monkey was removed from the LDL1 density subclass with a half-life of 5.5-10.3 h. Much of the radioactivity injected as LDL1 was converted to denser LDL (LDL2). Labeled LDL2 injected into the monkey was not converted to LDL1. Thus, at least two kinetically distinct subpopulations of LDL circulate in the plasma of this species. The lighter LDL is to a large extent a metabolic precursor of the more dense LDL (LDL2).     

Purine nucleotide catabolism in rat liver: labelling of uric acid and allantoin after administration of various labelled precursors

Uric acid and allantoin are the key compounds of purine nucleotide catabolism formed in liver and many other organs of the rat. We observed that, after administration of 14C-formate, incorporation of radioactivity into uric acid and allantoin is not similar, as one would expect. The phenomenon was demonstrated to be specific to liver and perfused liver, and not to other organs such as heart, jejunal mucosa, lung, spleen, and kidney. To interpret these results, the specific radioactivity of uric acid and allantoin in rat liver were analysed comparatively, after administration of the following labelled precursors: 14C-glycine, 14C-formate, 14C-hypoxanthine, 14C-uric acid and 14C-adenine. After administration of 14C-formate the specific radioactivity of allantoin was higher than that of uric acid and the same behavior was observed after 14C-uric acid and 14C-hypoxanthine, but not after 14C-glycine and 14C-adenine administration. The results indicate that the rate of their incorporation into uric acid and allantoin, and the subsequent export of these compounds into serum, can only partially explain the observed phenomenon, while the presence of different pools of uric acid and allantoin may give a complete explanation.     

Nicotinamide induces differentiation of embryonic stem cells into insulin-secreting cells

The poly(ADP-ribose) polymerase (PARP) inhibitor, nicotinamide, induces differentiation and maturation of fetal pancreatic cells. In addition, we have previously reported evidence that nicotinamide increases the insulin content of cells differentiated from embryonic stem (ES) cells, but the possibility of nicotinamide acting as a differentiating agent on its own has never been completely explored. Islet cell differentiation was studied by: (i) X-gal staining after neomycin selection; (ii) BrdU studies; (iii) single and double immunohistochemistry for insulin, C-peptide and Glut-2; (iv) insulin and C-peptide content and secretion assays; and (v) transplantation of differentiated cells, under the kidney capsule, into streptozotocin (STZ)-diabetic mice. Here we show that undifferentiated mouse ES cells treated with nicotinamide: (i) showed an 80% decrease in cell proliferation; (ii) co-expressed insulin, C-peptide and Glut-2; (iii) had values of insulin and C-peptide corresponding to 10% of normal mouse islets; (iv) released insulin and C-peptide in response to stimulatory glucose concentrations; and (v) after transplantation into diabetic mice, normalized blood glucose levels over 7 weeks. Our data indicate that nicotinamide decreases ES cell proliferation and induces differentiation into insulin-secreting cells. Both aspects are very important when thinking about cell therapy for the treatment of diabetes based on ES cells.