There is a futile cycle of pump and leak of protons across the mitochondrial inner membrane. The contribution of the proton cycle to standard metabolic rate is signi®cant, particularly in skeletal muscle, and it accounts for 20% or more of the resting respiration of a rat. The mechanism of the proton leak is uncertain: basal proton conductance is not a simple biophysical leak across the unmodi®ed phospholipid bilayer. Equally, the evidence that it is catalysed by homologues of the brown adipose uncoupling protein, UCP1, is weak. The yeast genome contains no clear UCP homologue but yeast mitochondria have normal basal proton conductance. UCP1 catalyses a regulated inducible proton conductance in brown adipose tissue and the possibility remains open that UCP2 and UCP3 have a similar role in other tissues, although this has yet to be demonstrated.

Early experiments indicated that islet beta-cells substantially metabolized L-alanine but that insulin secretion was largely unaffected by the amino acid. It was subsequently demonstrated using more intricate studies that L-alanine is a strong stimulus to insulin secretion in the presence of glucose in normal rodent islets and beta-cell lines. Using (13)C nuclear magnetic resonance (NMR), we have demonstrated substantial oxidative metabolism of L-alanine by the clonal beta-cell line BRIN-BD11, with time-dependent increases in production of cellular glutamate and aspartate. Stimulatory effects of L-alanine on insulin secretion were attenuated by the inhibition of beta-cell oxidative phosphorylation using oligomycin. Additionally, we detected substantial production of lactate, alanine, and glutamate from glucose (16.7 mmol/l) after 60 min. On addition of 10 mmol/l L-alanine to a stimulus of 16.7 mmol/l glucose, the utilization rate of glucose increased approximately 2.4-fold. L-Alanine dramatically enhanced NMR-measurable aspects of glucose metabolism (both oxidative and nonoxidative). The enhanced rate of entry of glucose-derived pyruvate into the tricarboxylic acid (TCA) cycle in the presence of alanine may have stimulated rates of generation of key metabolites, including ATP, which affect the insulin secretory process. Thus L-alanine metabolism, in addition to the enhancing effect on glucose metabolism, contributes to the stimulatory effects of this amino acid on insulin secretion in vitro.

Dynamic contrast agent-enhanced magnetic resonance imaging measurements of the perfusion of an immunogenic murine tumour showed that immune rejection was preceded by an increase in the apparent vascular volume of the tumour. This increase in vascularity, which has been observed previously in other tumours undergoing immune rejection, was confirmed by histological analysis of tumour sections obtained postmortem. Magnetic resonance imaging measurements similar to this could be used in the clinic to monitor the early responses of tumours to immunotherapy, before there is any change in tumour growth rate or volume.

No abstract

Much of the evidence supporting a mitochondrial uncoupling function for the homologues of UCPl (e.g. UCP2, UCP3) comes from experiments in which the levels of protein expression have been genetically manipulated. Here we present evidence, from yeast genetically modified to express these proteins, that these experimental manipulations can lead to an uncoupling of oxidative phosphorylation that does not represent a native activity of the protein. We used UCP1, whose native uncoupling activity can be fully inhibited by GDP, to demonstrate that artefactual uncoupling occurs at higher levels of protein expression. UCP3 similarly uncouples when expressed at these high levels. When we expressed UCP3 in yeast mitochondria at levels similar to, or 2-fold greater than, those found in mouse skeletal muscle (73 ? 53 ng / mg mitochondria1 protein) no uncoupling was observed. UCP3 only uncoupled at levels of expression that were 3-fold or more than in the mouse. This uncoupling was insensitive to GDP. The stimulation of uncoupling by palmitate in UCP1-expressing mitochondria was not duplicated in UCPZ or UCP3 expressors.42 Discriminatory antibody raised to UCP-3 used to detect protein in a number of systems The human UCP-3 (hUCP-3) and human UCP-2 (hUCP-2) proteins were overexpressed in the E. coli strain, BL21 pLysS using the expression vector PET 21d. mg amounts of U C P protein were expressed and purified in inclusion bodies to a high level (70%) of purity. Inclusion bodies were isolated by previously published protocol (Echtay et al,, 1999). Antibodies raised in rabbit to a hydrophilic peptide from rat UCP-3 (rUCP-3) protein (residues 149-163) were tested in this system. Western immunoblotting analysis, using the anti-UCP-3 peptide, detected E. coli expressed hUCP-3 but not hUCP-3 protein.Uncoupling protein 3 (UCP-3) is preferentially expressed in skeletal muscle (I).It has been shown in mice that low-intensity exercise bouts of 2 hourdday for 2 weeks produce increases in UCP-3 mRNA levels 3 hours post-exercise which decay to resting levels 24hours post-exercise (2). The aim of this study was to investigate the effects of endurance swimming training on UCP-3 protein expression in mouse skeletal muscle.Control animals performed no exercise while trained animals performed bouts of weighted exercise endurance training for up to 2 hours duration, 5 daydweek, for 17 weeks. 24hours following the training program animals were killed by C 0 2 asphyxiation,and the hind limb muscles soleus, extensor digitorum longus (EDL), plantaris and gastrocnemius were dissected free and intact.Muscle homogenates were prepared and analysed using western blot analysis for total UCP-3 protein content,corrected for mitochondrial number according to cytochrome-c content.Our data show an increase in UCP-3 protein expression in EDL and plantaris from trained mice compared with the control mice.As yet no detectable changes are seen in soleus and gastrocnemius.These results suggest that UCP-3 does play some as of yet undetermined role during endurance ex...