6.1. Presence and significance of brown adipose tissue

The results of the present study showed that the majority of adipose tissue in the newborn reindeer is functional BAT. The adipose tissue in the newborn reindeer showed the typical cell morphological features of BAT characterized in various species (Néchad 1986). The appearance and amount of mitochondria was paid particular attention, but the multilocularity of fat was not used as a criterion as it varies greatly in both BAT and WAT (Cannon & Nedergaard 1985, Néchad 1986, Trayhurn 1993). In addition to the high mitochondrial volume, evidence for the typical spot-like sympathetic nerves around adipocytes, for high aerobic capacity and for the presence of brown fat-specific UCP (UCP₁) all support the conclusion that most of the regularly prominent adipose tissue in the newborn reindeer represents active BAT. Altogether, the results support the conclusion that BAT has a fundamental significance for the NST of the newborn reindeer and their survival in the cold during close postnatal period.

BAT was present as distinct depots in more than ten different locations in the body, corresponding largely to the locations described in other newborn ruminants, including lambs (Gemmel et al. 1972, Alexander & Bell 1975), bovine calves (Alexander et al. 1975) and muskoxen (Blix et al. 1984) and other large precocial species (Néchad 1986). The strategic distribution of BAT within the main body cavities and close to vital organs and blood vessels supports the distribution of heat produced by BAT both locally and on a whole-body basis. The proportional size and importance of BAT depots varies greatly between species (Nedergaard et al. 1986). The newborn reindeer had a substantial depot of BAT in the perirenal-abdominal region (1/3 of all) which is typical for ruminants (Alexander & Bell 1975, Alexander et al. 1975). This depot is obviously one of the major sites of NST in newborn reindeer as shown by its highest aerobic capacity. The third largest BAT depot located on both sides of sternum beneath the pectoral muscles in the newborn reindeer has not previously been described in ruminants. All the depots classified as BAT occurred regularly in the data. However, the subcutaneous adipose tissue depots, which had no cellular morphology of BAT but resembled common WAT, were present only in some of the newborn reindeer.

Although BAT was the dominant type of adipose tissue in the newborn reindeer, its proportion of the body weight was only 1-2 %. This figure agrees with earlier findings in newborn ruminants and other large precocial species (Alexander & Bell 1975, Alexander et al. 1975). In small rodents such as guinea pigs, by contrast, the proportion of BAT can be 5 % of body weight (Néchad 1986). These species usually have considerably more WAT than ruminants. For comparison, the newborn human child has about 1-2 % of BAT but as much as 15-20 % of the WAT of their body weight (Lean & James 1986). In spite of the small amount of BAT, the thermogenesis of BAT contributes significantly to the metabolism and heat production of an animal. The thermogenic capacity of BAT (500 W·kg^-1) is about four times that of muscles and about 300 times that of other tissues (Girardier 1983). Thus, stimulation of 1 g of BAT can double the basal metabolic rate of a rat and 30-60 g of BAT can increase the basal metabolic rate of a human child by 110-170 % (Lean & James 1986). In newborn reindeer, NA-induced NST results in a three-fold increase in maximal heat production at +10°C (Soppela et al. 1986). The distribution of heat is intensified by the lively blood circulation of BAT (Alexander et al. 1973).

During the first weeks of life, the BAT of the newborn reindeer transformed into a tissue with the general histological characteristics of WAT. These changes included the disappearance of mitochondria and spot-like symphathetic innervation while the adipocytes accumulated lipids and became gradually unilocular (I). Simultaneously, the aerobic capacity of the tissue decreased. A similar histological development has been reported in the lamb (Gemmel et al. 1972) and the bovine calf (Alexander et al. 1975) and it appears characteristic of precocial species during the postnatal period in a natural environment (Nedergaard et al. 1986). The changes both at cellular and biochemical levels are strikingly matched with a fall in the capacity for NA-induced thermogenesis during the weeks of life in reindeer (Soppela et al. 1986). The reduction in the capacity for NST is likely to reflect a decrease in the demand for thermoregulatory heat production. With age, both insulation and the surface to volume ratio improve and this diminishes the demand for extra heat. The postnatal inactivation of BAT and its conversion to WAT-like tissue can be delayed or reversed by the stimulation of the tissue by its sympathetic innervation which can be activated by cold exposure (Gemmel et al. 1972).

As the newborn reindeer had only little WAT, but this tissue appeared during later life to the identical locations as BAT, it is possible that these two tissues have the same origin or that they develop from the same preadipocytes. They can thus represent different forms of the same tissue, as has been suggested in the goat (Trayhurn et al. 1993b). Very little is known about the ontogeny of interconversion of the two types of adipose tissues.

The immunoblotting studies indicated that almost all adipose tissues in the newborn reindeer had immunoreactivity consistent with UCP₁ and were thus ‘brown’ by their nature (II). The results are in line with those results obtained by conventional methods, except for the coronary adipose tissue which had no UCP₁ and resembled BAT only by its cellular morphology (I). Identified BAT depots in the reindeer calves agree with the depots identified by the presence of UCP₁ in bovine calves and lambs (Casteilla et al. 1987, 1989, Trayhurn et al. 1993a), and goat kids (Trayhurn et al. 1993b). However, fewer depots were reported in these species than in the reindeer. In the present study, there were no subcutaneous adipose tissue samples for the immunoblotting studies of UCP₁ as it was so rare (I). Subcutaneous depots have been judged ‘white’ based on the missing immunoreactivity for UCP₁ in the newborn calf and lamb (Casteilla et al. 1987) but ‘brown’ based on the existence of UCP₁ in lambs and kids (Trayhurn et al. 1993a, b). Such contradictory results suggest that the tissues were not necessarily homologous. It is also possible that the ‘phenotype’ of this tissue varies according to its functional requirements.

Coronary adipose tissue was the only depot in the newborn reindeer that did not display UCP_1, or the definite biochemical criteria of BAT. Therefore, this depot is probably not essential for thermogenesis. Distinct coronary WAT is also found in adult reindeer, and this tissue exists in significant amounts in the Svalbard reindeer (Pond et al. 1993). The very few previous studies of the cardiac adipose tissue in mammals (Marchington et al. 1989, Marchington & Pond 1990) have suggested that this depot may have a special function to fuel cardiac muscle and/or mop up dangerous excesses of fatty acids in the blood. Well-developed thoracic BAT is characteristic to hibernators (Néchad 1986) that need to warm the heart from a very low beat when arousing from hibernation.

Developmental studies indicate that UCP₁ is present in foetal reindeer by late gestation, about two weeks pre partum. However, the precise stage at which the protein appears in utero was not determined. UCP₁ is detectable in the bovine calf at 80 days pre partum and its expression increases toward the end of gestation (Casteilla et al. 1987, 1989). Foetal reindeer therefore clearly have the potential for BAT thermogenesis and the development of the tissue in utero presumably ensures that the calf is well prepared for the large change in ambient temperature (30-60°C) that occurs at birth. The results from the experiments in which perirenal adipose tissues of newborn red deer were probed for the mRNA for UCP₁ indicate that the gene coding for the protein is strongly expressed around the time of birth. This result is in line with the findings for other newborn ruminants (Casteilla et al. 1987, 1989, Trayhurn et al. 1993 a, b) and rabbit (Rozon et al. 1989).

UCP₁ was present in most adipose tissues of reindeer during the first days after birth but there was a rapid loss of protein thereafter and protein was not evident at the age of 2 months (II). Similar results have also been found in other ruminants (Casteilla et al. 1987, 1989, Trayhurn et al. 1993b). The disappearance of UCP₁ confirms that the histologically recognizable conversion of BAT to WAT-like tissue clearly occurs at the functional level. The rates of the postnatal disappearance of UCP₁ in different depots were not examined here in reindeer. In the goat kid, the disappearance of UCP₁ begins in subcutaneous adipose tissues (Trayhurn et al. 1993b). In the present study, the oldest animals were 6 month-old calves and adult reindeer that had undergone partial cold acclimatization during autumn and winter. No evidence for the presence of UCP₁ was found in their adipose tissues. This suggests that BAT does not reappear in older calves or in adult reindeer in natural conditions.

A prolonged, cold-mimicking β₃-adrenergic stimulation has not been found to re-induce UCP₁ in several adipose tissues of 1-year old reindeer calves although the sympathetic nervous system and lipolysis have been activated (Soppela, Trayhurn & Nieminen, unpublished observations). These findings suggest that there may be a strong inhibitory factor for UCP₁ gene expression in the adipose tissues of reindeer during winter that blocks the reconversion of BAT and dictates the ‘normal’ function of WAT. Reindeer are principally well adapted to even extreme cold due to their prime insulation (Scholander et al. 1950, Nilssen et al. 1984b) and their thermoregulatory costs appear to be substituted by heat generated from activity (Nilssen et al. 1984a). Therefore, reindeer do not probably need thermogenesis based on BAT, or such use of adipose tissues might be even wasteful. The reconvertibility of BAT in reindeer and similar large mammals in adult age is still an open question.

Anatomical and histological methods in the studies of BAT have been largely overruled by specific biochemical and molecular biological methods based on identifying UCP₁ and its mRNA. The present results show that active BAT can adequately be identified by a combination of electron and fluorescence microscopy supported by aerobic measurements. However, other, less active, fatty forms of BAT cannot be separated from WAT without the identifying UCP₁ or its mRNA (Trayhurn 1993). Accurate determination of the anatomical locations and the systematic description of adipose tissues is important for comparison of the biochemical properties of these tissues. This is particularly important in studies of adipose tissues which, exceptionally among the tissues of birds and mammals has largely missed the description and anatomical definitions that are foundation of comparative studies and the interpretation of any tissue (Wasserman 1965, Pond 1978, 1986, 1999).

One of the most interesting observations in the anatomy of the adipose tissues in reindeer was that the locations of BAT in the newborn reindeer and the locations of WAT in the older calves and adults were homologous. This supports the view of Lonèar (1991) that there may basically exist only one type of adipose tissue in large mammals, convertible adipose tissue (CAT) which is ‘brown’ in the newborns and ‘white’ in the older animals, depending on the biochemical characteristics of its mitochondria. However, it is important to note that adults may have also many other adipose tissue depots in addition to those present in newborns. At least adult semi-domesticated reindeer and Svalbard reindeer have many large superficial depots (Pond et al. 1993) that were not present here in the newborn reindeer.