John H. Beattie

Physiological role of adipose tissue: white adipose tissue as an endocrine and secretory organ

The traditional role attributed to white adipose tissue is energy storage, fatty acids being released when fuel is required. The metabolic role of white fat is, however, complex. For example, the tissue is needed for normal glucose homeostasis and a role in inflammatory processes has been proposed. A radical change in perspective followed the discovery of leptin; this critical hormone in energy balance is produced principally by white fat, giving the tissue an endocrine function. Leptin is one of a number of proteins secreted from white adipocytes, which include angiotensinogen, adipsin, acylation-stimulating protein, adiponectin, retinol-binding protein, tumour neorosis factor a, interleukin 6, plasminogen activator inhibitor-1 and tissue factor. Some of these proteins are inflammatory cytokines, some play a role in lipid metabolism, while others are involved in vascular haemostasis or the complement system. The effects of specific proteins maybe autocrine or paracrine, or the site of action maybe distant from adipose tissue. The most recently described adipocyte secretory proteins are fasting-induced adipose factor, a fibrinogen-angiopoietin-related protein, metallothionein and resistin. Resistin is an adipose tissue-specific factor which is reported to induce insulin resistance, linking diabetes to obesity. Metallothionein is a metal-binding and stress-response protein which may have an antioxidant role. The key challenges in establishing the secretory functions of white fat are to identify the complement of secreted proteins, to establish the role of each secreted protein, and to assess the pathophysiological consequences of changes in adipocyte protein production with alterations in adiposity (obesity, fasting, cachexia). There is already considerable evidence of links between increased production of some adipocyte factors and the metabolic and cardiovascular complications of obesity. In essence, white adipose tissue is a major secretory and endocrine organ involved in a range of functions beyond simple fat storage.

Induction, Regulation, Degradation, and Biological Significance of Mammalian Metallothioneins

MTs are small cysteine-rich metal-binding proteins found in many species and, although there are differences between them, it is of note that they have a great deal of sequence and structural homology. Mammalian MTs are 61 or 62 amino acid polypep-tides containing 20 conserved cysteine residues that underpin the binding of metals. The existence of MT across species is indicative of its biological demand, while the conservation of cysteines indicates that these are undoubtedly central to the function of this protein. Four MT isoforms have been found so far, MT-1, MT-2, MT-3, and MT-4, but these also have subtypes with 17 MT genes identified in man, of which 10 are known to be functional. Different cells express different MT isoforms with varying levels of expression perhaps as a result of the different function of each isoform. Even different metals induce and bind to MTs to different extents. Over 40 years of research into MT have yielded much information on this protein, but have failed to assign to it a definitive biological role. The fact that multiple MT isoforms exist, and the great variety of substances and agents that act as inducers, further complicates the search for the biological role of MTs. This article reviews the current knowledge on the biochemistry, induction, regulation, and degradation of this protein in mammals, with a particular emphasis on human MTs. It also considers the possible biological roles of this protein, which include participation in cell proliferation and apoptosis, homeostasis of essential metals, cellular free radical scavenging, and metal detoxification.

Copper and zinc metabolism in health and disease: speciation and interactions

It has long been evident that the metabolism of trace elements cannot be considered in isolation. A wide range of nutritional and physiological factors can influence their uptake, transport and storage, with subsequent enhancement of susceptibility to deficiency or toxicity states. Interactions occur with other trace elements and these can be classified as competitive or non-competitive, direct or indirect. However, their physiological or toxicological significance is sometimes debatable, partly because they were demonstrated under extreme experimental conditions where massive doses of the antagonistic metal were administered, often by parenteral routes and to animals whose trace element status was already severely compromised. Our understanding of the mechanisms of the interactions is frequently limited and indeed in only a few cases has it been possible to describe these on a molecular basis. It is almost traditional to cite the early work of Hill & Matrone (1970) as the first serious attempt to describe trace element interactions on a rational basis. They postulated that elements with similar physical or chemical properties will act antagonistically to each other biologically. The implication was that such metals could compete for binding sites on transport proteins or on enzymes requiring metals as co-factors. Over subsequent years, much evidence was produced in support of this view but rarely was the specific site of interaction identified, other than at tissue level. In part this reflected somewhat embarrassing limitations in our knowledge of the proteins involved in the intracellular and extracellular transport and storage of trace metals.

Characterization of metallothionein isoforms. Comparison of capillary zone electrophoresis with reversed-phase high-performance liquid chromatography

The purpose of this study was to compare and contrast the separation of metallothionein (MT) isoforms by reversed-phase high-performance liquid chromatography (RP-HPLC) with capillary zone electrophoresis (CZE). RP-HPLC was performed on a Vydac C8 column eluted with a linear acetonitrile gradient. CZE was performed in a 57 cm x 75 microns I.D. fused-silica tube at an operating voltage of 30 kV. Phosphate buffer (10 mM) at pH 2.5, 7.0 and 11.0 was used for both separations. CZE at pH 2.5 resolved three distinct peaks of rabbit liver MT which were incompletely resolved at pH 7.0 or 11.0. RP-HPLC at pH 2.5 gave two peaks and the resolution was not as good as with CZE at the same pH. At pH 7.0 or 11.0, RP-HPLC of rabbit liver MT gave a single predominant peak of unresolved MT-1 and MT-2. Purified rabbit liver MT-1 and MT-2 were used to verify the identity of these peaks. In contrast, MT from horse kidney demonstrated three predominant peaks which were best resolved by CZE at pH 11.0, whereas RP-HPLC resolved only two peaks at pH 11.0 and 7.0. At pH 2.5, RP-HPLC of horse kidney MT gave three peaks, though two of the peaks were incompletely separated. We conclude that pH has a considerable impact on the resolution of MT isoforms by CZE and RP-HPLC and that it is possible to exploit changes in pH to optimize the separation of isoforms for a particular species of MT. When samples of human and sheep liver MT-1, both of which exhibit microheterogeneity, were subjected to CZE, a single predominant peak was observed at each pH value. RP-HPLC of human liver MT-1 at pH 2.5 yielded two peaks that were incompletely resolved. Purified chick liver MT and rat liver MT-1 and MT-2 gave a single predominant peak at all pH values on CZE. In contrast, pig liver MT-1 and MT-2 each exhibited multiple peaks when subjected to CZE, the number of which depended on the pH used to separate the MT. In conclusion, CZE, with its orthogonal selectivity, and RP-HPLC make an excellent combination for the separation and characterization of MT isoforms. Because CZE is rapid (run times typically < 10 min) and requires little sample (< 100 nl), MT samples can readily be analyzed by CZE in conjunction with RP-HPLC or other techniques in order to maximize the information obtained about the individual isoforms.

SEPARATION OF METALLOTHIONEIN ISOFORMS BY CAPILLARY ZONE ELECTROPHORESIS

The potential of capillary zone electrophoresis (CZE) for the analysis of metallothionein (MT) isoforms was investigated. CZE was performed using two different systems, (1) a laboratory-constructed instrument with an ISCO UV detector and (2) a Waters Quanta 4000 system. Capillaries were of 75 microns I.D. x ca. 1 m in length and loading times were up to 40 s by gravity or 4 s by electrokinetic migration at 30 kV. Samples were dissolved in 10 mM Tris-HCl buffer, pH 9.1, and electrophoresis was performed at 30 kV using a 50 mM Tris-HCl, pH 9.1 running buffer. Detection was by UV absorbance at 185 or 214 nm. Purified and semipurified MT samples were analysed for qualitative assessment of purity, relative isoform abundance and separation characteristics of MT from different species. As progress towards the development of a quantitative assay, the linearity of calibration curves and simple methods of sample preparation for analysis by CZE were investigated. Complete separation of a mixture of the two major MT isoforms was achieved in less than 5 min and the technique was found to be very useful for qualitative analysis of MT. Using a rabbit liver MT standard (500 micrograms/ml-1), a linear relationship was found between the gravity load time and the integrated peak area. Standard calibration curves were also linear and the detection limit for both CZE instruments under our separation conditions was 1-10 micrograms MT ml-1. The successful use of two solvent extraction procedures for tissue samples demonstrated the potential of CZE for routine quantitative analysis of MT.

Lymphocyte metallothionein mRNA responds to marginal zinc intake in human volunteers.

Marginal Zn deficiency is thought to be prevalent in both developed and developing countries. However, the extent of Zn deficiency is not known, due to the lack of a reliable diagnostic indicator. Blood plasma and erythrocyte concentrations of metallothionein (MT) reflect Zn status, but measurement of MT is dependent on the availability of sensitive immunoassays. Our aim was to show whether measurement of T lymphocyte MT-2A mRNA, using a competitive reverse transcriptase (RT)--polymerase chain reaction (PCR) assay, could indicate Zn status in human subjects in a residential Zn-depletion study. In the study, the Zn intake of seven volunteers was maintained at 13.7 mg/d for 5 weeks (baseline) followed by 4.6 mg/d for 10 weeks (marginal intake) and then 13.7 mg/d (repletion) for 5 weeks. The quantitative assay was developed using standard techniques and concentrations of MT-2A mRNA were normalized by reference to beta-actin mRNA which was also measured by competitive RT--PCR assay. An alternative method of measuring the PCR product using capillary electrophoresis with laser-induced fluorescence detection was also evaluated. There was considerable inter-individual variation in MT-2A mRNA concentration and the mean level at the end of the baseline period was 10.3 (SE 3.7) fg MT-2A mRNA/pg beta-actin mRNA, which then decreased by 64 % during the low Zn intake period. After repletion, MT-2A mRNA returned to baseline concentrations. In contrast, plasma Zn was unchanged by marginal Zn intake or repletion. The effect of low Zn in all individuals was consistent. We conclude that this assay is a sensitive method of evaluating marginal changes in dietary Zn intake.

On-line solid-phase extraction-capillary electrophoresis for enhanced detection sensitivity and selectivity : application to the analysis of metallothionein isoforms in sheep fetal liver

A simple and rapid capillary electrophoresis (CE) method for analysis of metallothionein (MT) isoforms is described. A modified two-step solvent extraction procedure was used in combination with CE and an on-line solid-phase preconcentration device for sensitive and reproducible detection of MT isoforms in sheep fetal liver. Preparation of twenty samples was practicable within a working day with subsequent automated overnight analysis by solid-phase extraction (SPE)-CE. A commercially available divinylbenzene-based reversed-phase resin was found to be most suitable for the SPE device because it is resistant to extremes of pH and can be adequately regenerated between analyses. Each SPE device was readily constructed from commonly available materials and was used for the reproducible separation of over 100 biological samples before replacement. Precision of analysis within or between sample batches was < 10% and usually < 5% while detection limits were at least 28 ng/ml for standards and 272 ng/ml for biological samples. This would indicate a detection limit of about 0.5 microgram/g wet weight of tissue. Recovery of MT from tissue cytosols by solvent extraction was measured using radiolabeled MT and was found to be just over 50% increasing to almost 70% by addition of NaCl to the homogenisation buffer. The combined solvent extraction and SPE-CE methodology was applied to the analysis of MT in sheep fetal liver and the results compared favorably with those obtained by high-resolution chromatography. MT-1 levels were 2 to 4-times higher than those of MT-2 and both isoforms decreased from day 89 to day 136 of gestation. These results were compared with MT levels in fetal liver from sheep embryos that had been perturbed by temporary transfer to an advanced uterine environment. Hepatic MT levels at day 136 of gestation were 3 to 8-times higher than in normal fetal liver and significant differences were observed with both isoforms.

The influence of a low-boron diet and boron supplementation on bone, major mineral and sex steroid metabolism in postmenopausal women

An increase in dietary intake of B from 0.25 to 3.25 mg/d has been reported to increase plasma oestradiol and testosterone and decrease urinary Ca excretion in postmenopausal women. Consequently, it is suggested that the higher level of B intake could reduce bone loss associated with the menopause and cessation of ovarian function. The present study was designed to investigate the effect of a B supplement on bone mineral absorption and excretion, plasma sex steroid levels and urinary excretion of pyridinium crosslink markers of bone turnover in healthy postmenopausal volunteers. The women were accommodated in a metabolic unit, given a low-B diet (LBD; 0.33 mg/d) for 3 weeks and were asked to take a B supplement of 3 mg/d in addition to the LBD for a further 3 weeks. Changing B intake from 0.33 to 3.33 mg/d had no effect on minerals, steroids or crosslinks. However, the LBD appeared to induce hyperabsorption of Ca since positive Ca balances were found in combination with elevated urinary Ca excretion. This phenomenon may have inhibited or obscured any effect of B.

Separation of metallothionein isoforms by micellar electrokinetic capillary chromatography

Current techniques for the separation and quantification of metallothionein isoforms have limited value for routine analysis. Isoforms having a similar charge have been separated successfully using reversed-phase HPLC but this technique suffers from a slow sample turnover time. The use of the surfactant sodium dodecyl sulphate for the separation of metallothionein isoforms by micellar electrokinetic capillary chromatography (MECC) is described. The charge-different isoforms MT-1 and MT-2 from rats, rabbits and sheep were separated within 9-12 min. In addition, a varying degree of heterogeneity was observed in purified samples of human MT-1, rat MT-2, rabbit MT-1, rabbit MT-2 and sheep MT-1. The behaviour of chicken MT was different from that of any other species. The separation of sheep liver extracts indicated the potential of MECC as the basis for a quantitative assay for both charge-different and charge-similar metallothionein isoforms.

Analysis of metallothionein isoforms by capillary electrophoresis: optimisation of protein separation conditions using micellar electrokinetic capillary chromatography

Capillary electrophoresis (CE) techniques have been successfully applied to the separation of metallothionein (MT) isoforms and have proved to be rapid, practical and economical. Study of a variety of different electrolytes and capillaries has shown that electrolyte buffer composition and capillary wall surface modifications can have considerable influence on isoform separation and resolution. Ionic surfactants such as sodium dodecyl sulphate (SDS) form micelles at elevated concentrations and the partitioning of molecules between the hydrophobic micelle phase and the aqueous phase and their resulting migration in an electric field is the basis of the technique known as micellar electrokinetic capillary chromatography (MECC). In the present work, we have used sheep and rabbit MT to optimise MECC conditions for analysis of MT isoforms. Capillaries of 57 cm gave much better separations than shorter columns although analysis times were increased to about 12 min. Changing the buffer and SDS concentration or the pH affected the selectivity of isoform separation and up to 5 isoforms in sheep MT and 6 in rabbit MT were completely or partially resolved. Comparing different diameter capillaries we conclude that 25 microns I.D. columns give better separations than 50 or 75 microns I.D. columns although sensitivity is reduced by a factor of about 3 and 5, respectively. Using our MECC conditions, columns coated with C1 or C18 hydrophobic material were not found to be useful in improving MT separation or resolution although further evaluation of these columns is in progress.(ABSTRACT TRUNCATED AT 250 WORDS)