Metallothionein gene expression in brown adipose tissue.

Reduced environmental temperature triggers a range of behavioml and physiological responses to maintain body temperature. Brown adipose tissue (BAT) is a major site of thennoregulatory heat production [ 11, and during cold exposure thennogenesis in the tissue is rapidly stimulated, through the release of noradrenaline by the sympathetic nervous system. There is a substantially increased requirement for oxygen by BAT during thennogenesis and this may be accompanied by an increase in free radical generation. Metallothionein (MT) is a low molecular weight metal-binding protein which is induced directly by metals such as Zn, Cd and Cu, and by a wide range of stress factors which elevate for example, steroids, cytokines and catecholamines [2]. MT protein levels in the liver and kidney have also been shown to increase when rats are exposed to 4'C for 24 h [3, 41. Since hepatic MT levels increase on cold exposure and the protein has a proposed role as an antioxidant, we have investigated MT gene expression in BAT. Groups of 6 male Hooded Lister rats (-260 g body weight.) were maintained at 25'C (control), or exposed to 6'C for either 6 or 24 h. A further group at 25'C were injected S.C. with ZnClz at a dose of 10 mg Zn.Kg-' body weight, and killed 6 h after the injection. Liver, kidney, interscapular BAT and epididymal white adipose tissue were removed and snap-frozen in liquid nitrogen. Total RNA was extracted, separated by agarose gel electrophoresis and transferred to a nylon membrane by capillary blotting, as described elsewhere [5 ] . Detection of MT-1 mRNA was by chemiluminescence using a specific 28-mer antisense oligonucleotide probe [6] labelled with digoxigenin. 18s rRNA was also measured by the same technique using a 35-mer antisense oligonucleotide with a consensus sequence for a wide variety of species IS]. MT-1 protein in soluble tissue extracts was measured by radioimmunoassay [7]. Cold exposure caused a time-related increase in hepatic MT-1 gene expression; Zn injection, however, had a much more profound effect on liver MT-1 mRNA levels (Fig. 1A). In contrast, Zn had no effect on MT-1 gene expression in BAT, whereas cold exposure clearly induced transcription of this gene, the effect being greater at 24 h (Fig. 1B). Analysis of MT-1 protein was consistent with these results, and the Zn levels in BAT were unaffected by Zn injection but raised by cold exposure (data not shown). MT-1 mRNA levels in kidney showed a Similar response to cold exposure and Zn injection as those in the liver. None of the treatments induced MT-I expression in white adipose tissue (data not shown). Preliminary results suggest that MT-2 gene expression in the tissues analysed here parallels that of MT-1. A key finding of this study is that in contrast to cold exposure which has a marked and time-related effect on MT induction in BAT, Zn injection is without effect on MT expression in the tissue. In this respect, the response of BAT to MT inducers may have some similarity with the response of the brain, where significant induction by metals is only achieved by direct injection into the organ. In this case, metal uptake into the brain is limited by the blood-brain barrier whereas the uptake or endogenous synthesis of other potential inducers of MT may not be restricted in this way. Our conventional view of MT expression may therefore require some revision and tissues not known for their rapid upregulation of MT gene transcription may, in practice, be responsive to specific inducing agents. A Biochernicdl Society Trdnsactions ( 1 996) 24 1