Magnesium Research (1993) 6, 2, 135-143
Summary: The short term regulation of heat production in brown adipose tissue mitochondria (BAT) of acutely Mg-deficient rats was demonstrated by comparing several parameters of mitochondrial energization. Mg deficiency in vivo had absolutely no effect on the BAT uncoupling protein concentration (UCP) which was only modified by thermal conditions. The same high concentration was observed 10 d cold exposed control and Mg-deficient rats. Four days of warm re-exposure at thermal neutrality led to a moderate 26 per cent decrease with both diets which was not modified by cold stress for 1 h. Proton conductance, CmH+, and proton motive force, Δp, were calculated from membrane potential and respiration rate measurements. The same high level CmH+ was observed in cold exposed rats with both diets. Compared to warm re-exposed control rats, CmH+ was threefold higher in the corresponding Mg-deficient group which indicated a much lower masking of the proton channel of UCP with the Mg-deficient diet. This difference was not dependent on the presence of magnesium in vitro. The basal CmH+. independent of UCP, was not altered by magnesium deficiency. These results emphasize that acute regulation of thermogenic BAT activity through the masking and unmasking process is altered when magnesium supply is limited in vivo. Key words: Magnesium deficiency, brown adipose tissue, mitochondria, uncoupling protein, energization parameters, cold exposure.
Too few studies have investigated the role of magnesium in a cold environment1. Cold exposure markedly increases the magnesium requirement. Although long term tolerance to cold exposure (5° C) is good in chronically magnesium-deficient rats, response to more severe cold is impaired2. Some swelling of brown adipose tissue (BAT) mitochondria is observed in situ in acute magnesium deficiency possibly reflecting some modification of BAT thermogenesis3.
Magnesium is the second most abundant and ubiquitous intracellular metal and it activates many steps in the cellular metabolic pathways4. Mitochondria from liver, heart and skeletal muscle have been proposed as target cell constituents of magnesium deficiency in vivo leading to swelling and partial uncoupling of oxidative phosphorylation5,6 and consequently to disturbances of energy metabolism7. It is not established if these effects are the consequence of a direct action of magnesium deficiency on magnesium-dependent processes8-10 or are secondary to alterations of mitochondrial membranes11.
BAT in rodents is the major site in the generation of thermoregulatory heat by cold-induced non-shivering thermogenesis and diet-induced thermogenesis12-14. The main mechanism for thermogenesis in BAT is a proton leak across the inner mitochondrial membrane. The respiration is controlled by an uncoupling protein (UCP) located in the membrane which acts as a proton translocator15. The proton extrusion linked to substrate oxidation is dissipated to produce heat rather than used to generate a high proton motive force driving ATP synthesis.
The development of thermogenesis in BAT involves mainly the sympathetic system via the release of noradrenaline13. The proton conductance of BAT mitochondria is in part regulated by the synthesis of UCP (adaptative changes) and by the masking/unmasking of its proton channel (acute variations)16 , both being under sympathetic control. Several studies have attracted attention to the possibility of relationships between these modifications of thermogenic activity and modifications of BAT mitochondrial membrane composition17,18.
The aim of the present work was therefore to study the short term regulation of heat production in BAT mitochondria of acute magnesium-deficient rats which was demonstrated by comparing several parameters of mitochondrial energization, including membrane potential, proton motive force and effective proton conductance.
Animals and diets
Male Long-Evans rats reared at 23° C were randomly divided into magnesium-deficient and control groups at 5-6 weeks of age (130-150 g). They received semi-synthetic diets (casein 20 per cent, sucrose 70.5 per cent, corn oil 5 per cent, mineral mixture 3.5 per cent and vitamin mixture 1 per cent) ad libitum as previously described19. The magnesium content of the diets, determined by analysis, was 30 mg/kg (magnesium-deficient) and 960 mg/kg (control). Distilled water was provided ad libitum.
To stimulate BAT thermogenesis two groups of rats were exposed to cold (5° C) for 10 d before they were killed (control: C-Cold; magnesium-deficient: D-Cold).
After a 10 d period of cold exposure two groups of rats were warm re-exposed for 4 d at thermal neutrality (28° C) (control: C-Warm; magnesium-deficient: D-Warm). Acute unmasking of the uncoupling protein resulted from a 1 h cold stress at 5° C of warm re-exposed rats (control: C-Stress; magnesium-deficient: D-Stress).
All rats had the same 18 d nutritional period by the time they were killed. With such diets hypomagnesaemia was usually observed in magnesium-deficient rats (0.2-0.3 mM) compared to controls (0.8 mM)20.
Interscapular BAT was rapidly dissected out and used for the experiments.
Isolation of mitochondria
Mitochondria were isolated by the method of Cannon and Lindberg 21. After the final centrifugation, they were kept in 250 mM sucrose, 5 mM TES, pH 7.2.
Oxygen consumption and membrane potential were determined simultaneously. Oxygen uptake was measured polarographically with a Clark-type electrode, and membrane potential with a laboratory constructed tetraphenylphosphonium (TPP+) selective electrode22 . Measurements were carried out in a medium containing 100 mM sucrose, 20 mM TES, 4 mM K2HPO4, 2 mM MgCl2, 1 mM EGTA, 1 per cent W/V fattyacid-free serum albumin, 5 µm rotenone (final volume 1.5 ml); 0.5 mg mitochondrial protein was added per assay. Respiration rate was modulated by varying the α-glycerophosphate substrate concentration.
Membrane potential was corrected to take into account the activity coefficient of TPP+ in the matrix according to Rottenberg23 (about 50 mV in these experimental conditions).
To calculate the proton conductance of the inner membrane CmH+ (nmol protons per mg protein flowing through the membrane per minute per mV of proton motive force, Δp), proton current JH+ was calculated from the respiratory rate. JO, on the assumption that six protons were extruded by the respiratory chain per two electrons transferred to oxygen (α-glycerophosphate oxidation). CmH+ was calculated according to the following equation:
where Jo was expressed in nat O per min per mg protein, and Δp=ΔΨ-59ΔpH was expressed in mV at 25° C.
Δp was calculated according to the linear relationship previously established experimentally between ΔΨ and Δp (in these conditions: 59ΔpH = 0.73ΔΨ -76 (in mV). Technical conditions applied here to calculate CmH+ have been discussed in detail elsewhere 17,24.
Uncoupling protein determination
Western blots were used. Rat mitochondria proteins were separated by polyacrylamide gel electrophoresis and electroeluted from the gels to nitrocellulose. Activity staining was effected with anti-ewe IgG conjugated to alkaline phosphatase to develop colouration. Densitometric scanning of blots were performed with a Shimazu CS930 densitometer25,26.
Mitochondrial proteins were assayed by the method of Lowry et al.27.
Data are expressed as mean ± SEM. The significance of the differences between different groups was analysed using Student's t test.
In magnesium-deficient rats the classical clinical symptoms of magnesium deficiency were observed. Hyperaemia of the ears occurred during the experimental period. Plasma magnesium concentration decreased about by 0.4 mM. Magnesium-deficient rats were notably hyperexcitable and showed significant growth retardation (about 10 per cent). Cold tolerance at 5° C was not affected by magnesium deficiency and body temperature was the same as in control rats.
Comparison of uncoupling protein content estimated by densitometric tracing of immunological blots indicated the same concentration in BAT mitochondria of 10 d cold-exposed control and magnesium -deficient rats (Fig. 1). Four days of warm re-exposure at thermal neutrality led to the same moderate decrease (26 per cent) with both diets. Cold stress for 1 h in warm re-exposed rats did not induce any UCP synthesis as previously seen24. Thus magnesium deficiency had absolutely no effect on UCP concentration, which was only modified here by thermal conditions.
Representative experiments in which values of respiration rate were plotted against values of membrane potential (flux/force relationships) are shown on Fig. 2. Respiration rate (and thus proton current) was modulated by varying the aglycerophosphate concentration (0.1-10 mM) in the absence of Ca2+. The same lower membrane potential was observed over the whole range of respiration rate in BAT mitochondria of 10 d cold exposed control and magnesium-deficient rats, indicating a high level of uncoupling. Four days of warm re-exposure greatly increased membrane potential in rats fed the control diet and to a much lower extent in the magnesium-deficient group. Acute exposure to cold of warm re-exposed rats, which partially unmasked the proton channel of UCP18, led to a decrease of membrane potential both in control and magnesium-deficient rats. Membrane potential was slightly lower in the deficient group.
From membrane potential and respiration rate, it is possible to calculate proton conductance, CmH+, and proton motive force, Δp (as described in Material and Methods). Figure 3 permits comparison of CmH+ over the whole range of Δp. The same high level of CmH+ was observed in cold-exposed rats with both diets. This comparable high level of CmH+ gives biochemical support to the fairly good cold tolerance of magnesium-deficient rats at 5° C previously observed by Heroux et al. 2. In warm re-exposed rats CmH+ was very low except at higher values of Δp where it was strongly enhanced. Between 130 and 180 mV, where comparison proved practicable, CmH+ was one order of magnitude higher in 10 d cold exposed control rats than in warm reexposed ones. Compared to warm re-exposed control rats, CmH+ was threefold higher in the corresponding magnesium-deficient group. As UCP content was the same in the two groups, this indicated a much lower masking of the proton channel of UCP with the magnesium-deficient diet. In the cold stressed groups CmH+ level was about half that observed in 10 d cold exposed rats.
Effect of magnesium in vitro
Figure 4 draws a comparison of respiratory studies in media with and without magnesium. In the absence of GDP, lack of magnesium in the respiratory medium led to the same decrease of membrane potential (about 8 mV) over the whole range of respiratory rate in BAT mitochondria of warm re-exposed control and magnesium-deficient rats. Thus the difference observed between these two groups is not dependent on the presence of magnesium in vitro. With UCP completely blocked by an optimal concentration of GDP17, membrane potential was increased. Superimposable force/flux relationships were observed in the two groups of rats, indicating that the basal proton conductance, independent of UCP, was not altered by magnesium deficiency. In presence of GDP, lack of magnesium in vitro decreased membrane potential to the same extent in the two groups (about 12 mV).
Our results clearly show an incomplete masking of the UCP-dependent proton conductance after recovery at thermal neutrality of 10 d cold exposed magnesium-deficient rats. On the other hand, the basal conductance measured in the presence of GDP, which completely block, UCP, was the same in all groups.
CmH+ is the primary functional parameter of UCP, the function of which is to channel protons9. Non-phosphorylating mitochondria were investigated in this study. Being dependent on the native proton conductance, state 4 is the most valuable criterion to assay proton leak associated with BAT thermogenic activity.
There is evidence from the literature that magnesium deficiency in vivo can disturb neural or hormonal influences such as catecholamines and insulin28.29 which have a primary role in or participate in the control of BAT thermogenesis13. However the possibility that the partial unmasking observed in BAT of warm re-exposed magnesium-deficient rats is due to some sympathetic stimulation via the release of noradrenaline is unlikely since the UCP content, which also depends of sympathetic stimulation, was the same as in controls. Moreover, in rats kept at 23° C no such impaired masking was observed: proton conductance of control and magnesium-deficient rats was similar (results not shown).
Based on UCP content and GDP binding in the regulatory site measurements, masking/unmasking of GDP binding is a well known phenomenon18 accompanied by large variations of the proton conductance which is the primary functional parameter of the protein16,18. During cold exposure, short term changes in BAT thermogenic activity are due more to unmasking of sites than to de novo synthesis of the uncoupling protein. Long term changes are, conversely, more likely to be due to an altered concentration of the protein. in vitro studies suggest a possible link between magnesium, affinity of nucleotides, and unmasking of UCP nucleotide regulatory sites (review in 1). In various tissues, several investigations have suggested mitochondrial dysfunction in vitro during magnesium deficiency in vivo. This includes loss of potassium and accumulation of calcium and sodium30. A partial uncoupling of oxidative phosphorylation indicated by lower ADP : O values is also observed in cardiac and hepatic mitochondria5,31.
BAT mitochondria exhibit remarkable plasticity. Ultrastructural changes have been observed during both acclimation and deacclimation to cold of control rats, these modifications being quite likely to be associated with modified ion distribution. Cold exposure is known to induce depletion of K+ and Mg2+ 3,32. Acute cold exposure or the short term effect of noradrenaline induces a rapid increase in respiration rate concurrently with reversible modifications in the morphology of BAT mitochondria such reorientation of cristae and enhanced mitochondrial volume3,32. These changes persist during the isolation of these mitochondria. Thus unmasking of the nucleotide regulatory site and proton channel of UCP is concomitant with ultrastructural changes which possibly lead to variable accessibility of the proton channel.
In the same way, in BAT of magnesium-deficient rats some swelling of mitochondria induce by Ionic modifications has been observed in situ3. It is possible that such modifications disturb the masking process.
Warm re-exposed control rats (after 10 days at 5° C) show great potential for UCP masking/unmasking which corresponds to variation of proton conductance over one order of magnitude: proton conductance is the same as for rats reared at thermal neutrality but there is 2.3 times more uncoupling protein18. Thus, in BAT of magnesium-deficient rats it is likely that the swelling of mitochondria observed in situ prevents the complete masking of the proton channel. In rats kept at 23° C, which show only a limited twofold amplitude for the masking process, no such difference was observed.
Several mitochondrial functions are magnesium-dependent. In addition to the well known magnesium activation of ATPase10, recent studies have identified several magnesium-dependent ion transports (uniports or antiports) present in mitochondria of various tissues including BAT8,9. Assumptions have been made that these transports, modulated in vitro by mitochondrial magnesium content, participate in the physiological homeostatic control of mitochondrial volume in vivo. Thus the partial masking of UCP observed here could be the direct physiological consequence of the alterations of magnesium-related activities.
During previous cold exposure in rats, BAT mitochondria experience large increases in oxygen consumption and thus oxygen radical generation which may be compensated by increases in antioxidant enzymes, ascorbate, and reduced glutathione33. Recent studies suggest that magnesium deprivation decreases membrane lipid protection against free radical injury . Thus increased peroxidation of the phospholipid environment of UCP, modifying the masking/unmasking process in BAT of magnesium-deficient rats, is another alternative possibility.
The mechanisms involved in the activation/inactivation of the proton channel by masking/unmasking of UCP have not been established. However, it is likely that some relationships exist between that process and modifications of membrane lipid composition which are induced by modulation of temperature exposure and sympathetic activity18,35.
Magnesium deficiency affects lipid metabolism36. In the inner mitochondrial membrane lipid region, both the phospholipid classes and their fatty acid components are modified by magnesium deficiency in hepatic and cardiac mitochondria and this may affect membrane function11. Thus, another possibility is that impaired masking observed here could be a secondary response induced by modifications of mitochondrial membrane composition.
In conclusion these results are the first answer to recent in vitro studies which suggest that magnesium status may intervene through different mechanisms in the regulation of BAT mitochondrial heat production (review in 1). They emphasize that the short term regulation of BAT thermogenic activity through the masking/unmasking process is altered when magnesium supply is modified in vivo.
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