Effect of a tumour-derived lipid-mobilising factor on glucose and lipid metabolism in vivo

Treatment of ex-breeder male NMRI mice with lipid mobilising factor isolated from the urine of cachectic cancer patients, caused a significant increase in glucose oxidation to CO2, compared with control mice receiving phosphate buffered saline. Glucose utilisation by various tissues was determined by the 2-deoxyglucose tracer technique and shown to be elevated in brain, heart, brown adipose tissue and gastrocnemius muscle. The tissue glucose metabolic rate was increased almost three-fold in brain, accounting for the ability of lipid mobilising factor to decrease blood glucose levels. Lipid mobilising factor also increased overall lipid oxidation, as determined by the production of 14CO2 from [14C carboxy] triolein, being 67% greater than phosphate buffered saline controls over a 24 h period. There was a significant increase in [14C] lipid accumulation in plasma, liver and white and brown adipose tissue after administration of lipid mobilising factor. These results suggest that changes in carbohydrate metabolism and loss of adipose tissue, together with an increased whole body fatty acid oxidation in cachectic cancer patients, may arise from tumour production of lipid mobilising factor. © 2002 Cancer Research UK.

In vivo vitamin C supplementation increases phosphoinositol transfer protein expression in peripheral blood mononuclear cells from healthy individuals

Ascorbate can act as both a reducing and oxidising agent in vitro depending on its environment. It can modulate the intracellular redox environment of cells and therefore is predicted to modulate thiol-dependent cell signalling and gene expression pathways. Using proteomic analysis of vitamin C-treated T cells in vitro, we have previously reported changes in expression of five functional protein groups associated with signalling, carbohydrate metabolism, apoptosis, transcription and immune function. The increased expression of the signalling molecule phosphatidylinositol transfer protein (PITP) was also confirmed using Western blotting. Herein, we have compared protein changes elicited by ascorbate in vitro, with the effect of ascorbate on plasma potassium levels, on peripheral blood mononuclear cell (PBMC) apoptosis and PITP expression, in patients supplemented with vitamin C (0-2 g/d) for up to 10 weeks to investigate whether in vitro model systems are predictive of in vivo effects. PITP varied in expression widely between subjects at all time-points analysed but was increased by supplementation with 2 g ascorbate/d after 5 and 10 weeks. No effects on plasma potassium levels were observed in supplemented subjects despite a reduction of K+ channel proteins in ascorbate-treated T cells in vitro. Similarly, no effect of vitamin C supplementation on PBMC apoptosis was observed, whilst ascorbate decreased expression of caspase 3 recruitment domain protein in vitro. These data provide one of the first demonstrations that proteomics may be valuable in developing predictive markers of nutrient effects in vivo and may identify novel pathways for studying mechanisms of action in vivo.

Structure and activity in assessing antioxidant activity in vitro and in vivo:a critical appraisal illustrated with the flavonoids

Structure–activity relationships are indispensable to identify the most optimal antioxidants. The advantages of in vitro over in vivo experiments for obtaining these relationships are, that the structure is better defined in vitro, since less metabolism takes place. It is also the case that the concentration, a parameter that is directly linked to activity, is more accurately controlled. Moreover, the reactions that occur in vivo, including feed-back mechanisms, are often too multi-faceted and diverse to be compensated for during the assessment of a single structure–activity relationship. Pitfalls of in vitro antioxidant research include: (i) by definition, antioxidants are not stable and substantial amounts of oxidation products are formed and (ii) during the scavenging of reactive species, reaction products of the antioxidants accumulate. Another problem is that the maintenance of a defined concentration of antioxidants is subject to processes such as oxidation and the formation of reaction products during the actual antioxidant reaction, as well as the compartmentalization of the antioxidant and the reactive species in the in vitro test system. So determinations of in vitro structure-activity relationships are subject to many competing variables and they should always be evaluated critically. (c) 2005 Published by Elsevier B.V.

The use of proteomic techniques to explore the holistic effects of nutrients in vivo

The availability of ‘omics’ technologies is transforming scientific approaches to physiological problems from a reductionist viewpoint to that of a holistic viewpoint. This is of profound importance in nutrition, since the integration of multiple systems at the level of gene expression on the synthetic side through to metabolic enzyme activity on the degradative side combine to govern nutrient availability to tissues. Protein activity is central to the process of nutrition from the initial absorption of nutrients via uptake carriers in the gut, through to distribution and transport in the blood, metabolism by degradative enzymes in tissues and excretion through renal tubule exchange proteins. Therefore, the global profiling of the proteome, defined as the entire protein complement of the genome expressed in a particular cell or organ, or in plasma or serum at a particular time, offers the potential for identification of important biomarkers of nutritional state that respond to alterations in diet. The present review considers the published evidence of nutritional modulation of the proteome in vivo which has expanded exponentially over the last 3 years. It highlights some of the challenges faced by researchers using proteomic approaches to understand the interactions of diet with genomic and metabolic–phenotypic variables in normal populations.

Tetrahydrobiopterin metabolism in depression and senile dementia of Alzheimer type

Excretion of biopterin and the related pteridines neopterin and pterin was measured in urine samples from a group of 76 male and female unipolar and bipolar depressed outpatients receiving lithium therapy, and compared to 61 male and female control subjects. The ratio of neopterin to biopterin excreted (N/B) was significantly higher in the patients than the controls. The significant positive correlation between urinary neopterin and biopterin shown by the controls was absent in the patients, indicating disrupted biosynthesis of tetrahydrobiopterin.Urinary cortisol excretion in depressed patients was similar to controls, implying normal hypothalmus-pituitary-adrenal axis function in these patients, Serum folate was shown to correlate with urinary total biopterin excretion in female unipolar patients. Two groups of elderly females with senile dementia of Alzheimer type (SDAT) were examined for urinary pteridine excretion. In the first study of 10 patients, the N/B ratio was significantly higher than in 24 controls and the ratio B/B+ N significantly lower. A second study of 30 SDAT patients and 21 controls confirmed these findings. However, neopterin correlated with biopterin in both patients and controls, indicating that the alteration in tetrahydrobiopterin metabolism may be different to that shown in depression. Lithium had no effect in vivo or in vitro on Wistar rat brain or liver biosynthesis of tetrahydrobiopterin at a range of concentrations and duration of dosing period, showing that lithium was not responsible for the lowered biopterin excretion by depressed patients. No significant effects on tetrahydrobiopterin metabolism in the rat were shown by the tricyclic antidepressant imipramine, the anticonvulsant sodium valproate, the vitamin folic acid, the anticatecholaminergic agent amethylparatyrosine, the synthetic corticosteroid dexamethasone, or stimulation of natural cortisol by immobilisation stress. Scopolamine, an ant ichol inergic drug, lowered rat brain pterin which may relate to the tetrahydrobiopterin deficits shown in SDAT.

The role of metabolism in the toxicity of N-alkylformamides

Using ionspray tandem mass spectrometry the glutathione conjugate SMG was identified as a biliary metabolite of DMF in rats (0.003% of a dose of 5OOmg/kg DMF i.p.). Formation of this metabolite was increased five fold after induction of CYP2E1 by acetone, and was inhibited to 20% of control values following pretreatment with disulfrram. Generation of SMG from DMF in vivo was shown to exhibit a large kinetic deuterium isotope effect (KWKD=10.1 ± 1.3), which most likely represents the product of 2 discrete isotope effects on N-demethylation and formyl oxidation reactions.The industrial solvent N,N-dimethylformamide (DMF) and the investigational anti-tumour agent N-methylformamide (NMF) cause liver damage in rodents and humans. The hepatotoxicity of N-alkylformamides is linked to their metabolism to N-alkylcarbamic acid thioesters. The enzymatic details of this pathway were investigated. Hepatocytes isolated from BALB/c mice which had been pretreated with acetone, an inducer of the cytochrome P-450 isozyme CYP2E1, were incubated with NMF (10mM). NMF caused extensive toxicity (> 90% ) as determined by lactate dehydrogenase (LDH) release, compared to cells from untreated animals. Incubation of liver cells with NMF for 6 hrs caused 60±17% LDH release whilst in the presence of DMSO (10mM), an alternative substrate for CYP2E1, LDH release was reduced to 20±10% . The metabolism of NMF to S-(N-methylcarbamoyl)glutathione (SMG) was measured in incubates with liver microsomes from mice, rats or humans. Metabolism of NMF was elevated in microsomes isolated from rats and mice pretreated with acetone, by 339% and 183% respectively. Pretreatment of animals with 4-methylpyrazole induced the metabolism of NMF to 280% by rat microsomes, but was without effect on NMF metabolism by mouse microsomes. The CYP2E1 inhibitors or alternative substrates diethyl dithiocarbamate (DEDTC), p-nitrophenol (PNP) and dimethyl sulphoxide (DMSO) strongly inhibited the metabolism of NMF in suspensions of rat liver microsomes, at concentrations which did not effect aminopyrine N-demethylation. The rate of metabolism of NMF to SMG in human microsomes correlated (r> 0.8) with the rate of metabolism of chlorzoxazone, a CYP2E1 probe. A polyclonal antibody against rat CYP2E1 (10mg/nmol P-450) inhibited NMF metabolism in microsomes from rats and humans by 75% and 80% , respectively. The amount of immunoblottable enzyme in human microsomes, determined using an anti-rat CYP2E1 antibody, correlated with the rate of NMF metabolism (r> 0.8). Purified rat CYP2E1 catalysed the generation of SMG from NMF. Formation of the DMF metabolite N-hydroxymethyl-N-methylformamide (HMMF) in incubations with rat liver microsomes was elevated by 200% following pretreatment of animals with acetone. Co-incubation with DEDTC (100μM) inhibited HMMF generation from DMF by 88% . Co-incubation of DMF (10mM) with NMF (1mM) inhibited the formation of SMG by 95% . A polyclonal antibody against rat CYP2E1 (10mg/nmol P-450) inhibited generation of HMMF in incubates with rat and human liver microsomes by 68.4% and 67.5% , respectively. Purified rat CYP2E1 catalysed the generation of HMMF from DMF. Using ionspray tandem mass spectrometry the glutathione conjugate SMG was identified as a biliary metabolite of DMF in rats (0.003% of a dose of 5OOmg/kg DMF i.p.). Formation of this metabolite was increased five fold after induction of CYP2E1 by acetone, and was inhibited to 20% of control values following pretreatment with disulfrram. Generation of SMG from DMF in vivo was shown to exhibit a large kinetic deuterium isotope effect (KHKD=10.1 ± 1.3), which most likely represents the product of 2 discrete isotope effects on N-demethylation and formyl oxidation reactions.

Tetrahydrobiopterin metabolism in dementia

The aim of this study was to establish levels of the enzymes involved in tetrahydrobiopterin (BH4) metabolism in human and rat brain preparations; to determine whether BH4 metabolism is altered in dementia, particularly in relation to senile dementia of the Alzheimer type (SDAT); and to examine the effect of aluminium on BH4 metabolism. Overall BH4 synthesis and dihydropteridine reductase (DHPR) activity were greater in the locus coeruleus than in the neocortex of elderly subjects. Sepiapterin reductase and DHPR activity showed a linear correlation with age in the temporal cortex. DHPR activity in the frontal cortex was relatively constant until the mid 60s and then fell with age. Overall BH4 synthesis showed a non-significant decline in temporal cortex and was significantly reduced in locus coeruleus preparations from SDAT subjects compared to control subjects. As DHPR, sepiapterin reductase and GTP cyclohydrolase activity were unaltered in SDAT we suggested that there is a lesion on the biosynthetic pathway between dihydroneopterin in triphosphate and BH4 in SDAT, possibly at the level of 6-pyruvoyl tetrahydropterin synthase. DHPR activity and BH4 synthesis capacity were unaltered in temporal cortex preparations from Huntingdon's disease subjects indicating that the defect in BH4 metabolism in SDAT is specific to the disease process and not a secondary consequence of dementia. The implications of altered BH4 metabolism in ageing and dementia are discussed. BH4 metabolism was examined in temporal and frontal cortex preparations from 4 subjects who had received peritoneal dialysis treatment. All patients had elevated serum aluminium levels. The data suggests that aluminium may inhibit DHPR activity in the frontal cortex resulting in diminished BH4 levels in the cells which leads to a compensatory increase in the activity of the biosynthetic pathway. Aluminium reversibly inhibited sepiapterin reductase activity in rat brain preparations but did not alter sepiapterin reductase activity in vivo. Overall BH4 synthesis and OTP cyclohydrolase activity were not affected by aluminium in vitro. The biosynthetic pathway was unaltered in rat brain preparations from animals receiving aluminium orally compared to control animals. DHPR activity was unaltered or increased in rat brain preparations from aluminium treated rats compared to the control group.

Changes in gene expression and morphology of mouse embryonic stem cells on differentiation into insulin-producing cells in vitro and in vivo

Background Embryonic stem (ES) cells have the potential to produce unlimited numbers of surrogate insulin-producing cells for cell replacement therapy of type I diabetes mellitus. The impact of the in vivo environment on mouse ES cell differentiation towards insulin-producing cells was analysed morphologically after implantation. Methods ES cells differentiated in vitro into insulin-producing cells according to the Lumelsky protocol or a new four-stage differentiation protocol were analysed morphologically before and after implantation for gene expression by in situ reverse transcription polymerase chain reaction and protein expression by immunohistochemistry and ultrastructural analysis. Results In comparison with nestin positive ES cells developed according to the reference protocol, the number of ES cells differentiated with the four-stage protocol increased under in vivo conditions upon morphological analysis. The cells exhibited, in comparison to the in vitro situation, increased gene and protein expression of Pdx1, insulin, islet amyloid polypeptide (IAPP), the GLUT2 glucose transporter and glucokinase, which are functional markers for glucose-induced insulin secretion of pancreatic beta cells. Renal sub-capsular implantation of ES cells with a higher degree of differentiation achieved by in vitro differentiation with a four-stage protocol enabled further significant maturation for the beta-cell-specific markers, insulin and the co-stored IAPP as well as the glucose recognition structures. in contrast, further in vivo differentiation was not achieved with cells differentiated in vitro by the reference protocol. Conclusions A sufficient degree of in vitro differentiation is an essential prerequisite for further substantial maturation in a beta-cell-specific way in vivo, supported by cell-cell contacts and vascularisation. Copyright (c) 2009 John Wiley & Sons, Ltd.