Our previous finding of a reduced energy metabolism in slow- and fast-twitch skeletal muscle fibres from the murine model of Duchenne muscular dystrophy (the mdx mouse) led us to examine the importance of intracellular glucose availability for a normal energy turnover. To this end, basal and KCl-stimulated (20.9 mM total extracellular K+) rates of glucose uptake (GUP) and heat production were measured in isolated, glucose-incubated (5 mM) soleus and extensor digitorum longus muscles from mdx and control C57B1/10 mice, in the presence and in the absence of insulin (1.7 nM). Under all conditions and for both muscle types, glucose uptake values for mdx and control muscles were similar although heat production was lower in mdx muscles. The marked stimulation of GUP by insulin in both mdx and control muscles had only minor effects on heat production. In contrast, glucose deprivation or inhibition of glycolysis with 2-deoxy-D-glucose (5 mM) significantly decreased heat production in control muscles only, which attenuated, although did not suppress, the difference in basal heat production between mdx and control muscles. Stimulation of heat production by a short-chain fatty acid salt (octanoate, 2 mM) was significantly less marked in mdx than in control muscles. Increased cytoplasmic synthesis of CoA by addition of 5 mM pantothenate (vitamin B5) increased the thermogenic response to glucose more in mdx than in control muscles. We conclude that the low energy turnover in mdx-mouse muscle fibres is not due to a decrease of intracellular glucose availability, but rather to a decreased oxidative utilization of glucose and free fatty acids. We suggest that some enzyme complex of the tricarboxylic acid cycle or inefficiency of CoA transport in the mitochondria could be involved.

SUMMARY1. The resting heat production rate (E) of soleus muscles from young rats and brown adipose tissue from adult rats was measured by means of a perfusable heat flux microcalorimeter in the absence and presence of ouabain. In the soleus muscle, the acute response of E to ouabain was compared with the ouabain-suppressible components of 22Na-efflux and 42K-influx.2. In standard Krebs-Ringer bicarbonate buffer, ouabain (10-3M) induced an immediate but transient decrease in E of around 5 %. Both in muscle and adipose tissue this was followed by a progressive rise in heat production rate.3. When the medium was enriched with Mg (10 mM), ouabain produced a sustained decrease in E of the same magnitude as in the standard medium and the secondary rise was less marked or abolished. Under these conditions, in the soleus muscle, ouabain inhibited E? by 5 % (i.e. by 1-76 + 0-22 mcal.g wet wt.-'.min-'), 22Na-efflux by 58 % (0.187 + 0.013,mole. g wet wt. -. min-') and 42K-influx by 34 % (0.132 ± 0028 molel. g wet w.l.min-1).4. When the muscles were loaded with Na by pre-incubation in K-free Mg-enriched medium, the addition of K (3 mM) induced an immediate ouabain-suppressible increase in E of 2 98 + 0 33 mcal. g wet wt.-. minand a concomitant stimulation of 22Na-efflux of 0-388 + 0 136 molel. g wet wt.-. min-.5. Maximum Na/ATP ratios for the active Na-K transport process were computed, with no assumption as to the in vivo free energy of ATP hydrolysis. These were 2 1, 1 9 and 2-3 under the conditions described in paragraphs (2), (3) and (4) respectively. 44A. CHINET, T. CLAUSEN AND L. GIRARDIER 6. The calculated reversible thermodynamic work associated with active Na-K transport corresponded to 34 % of the measured ouabaininduced decrease in P. On the premise that the maximum efficiency of the cellular energy conservation processes is 65 %, this estimate indicates that the minimum energetic efficiency of ATP utilization by the active Na-K transport process in mammalian muscle is 52 %.

SUMMARY1. The rate of energy expended for the clearance of sarcoplasmic Ca2+ by sarcoreticular Ca2+ uptake processess, plus the concomitant metabolic reactions, was evaluated from measurements of resting heat production by mouse soleus muscle before and after indirect inhibition of Ca2+ uptake by sarcoplasmic reticulum (SR).2. Direct inhibition of the Ca2+, Mg2+-ATPase of SR membrane in intact muscle preparations exposed to the specific inhibitor 2,5-di(tert-butyl-1 ,4-benzohydroquinone (tBuBHQ) slowly increased the rate of heat production (E). Indirect inhibition of SR Ca2+ uptake was obtained by reducing sarcoplasmic Ca2+ concentration (Ca2+) as a consequence of reducing Ca2+ release from the SR using dantrolene sodium. This promptly decreased E by 12 %. Exposure of the preparations to an Mg2+-enriched environment (high Mg2+) or to the chemical phosphatase 2,3-butanedione monoxime (BDM), two other procedures aimed at decreasing SR Ca2+ release, also acutely decreased E, by 20 and 24 %, respectively.3. Subthreshold-for-contracture depolarization of the sarcolemma achieved by increasing extracellular K+ concentration to 11-8 mm induced a biphasic increase of E: an initial peak to 290 % of basal E, followed by a plateau phase at 140 % of basal E during which resting muscle tension was increased by less than 3 %. Most, if not all, of the plateau-phase metabolic response was quickly suppressed by dantrolene or high Mg2+ or BDM. Another means of increasing SR Ca2+ cycling was to partially remove the calmodulin-dependent control of SR Ca2+ release using the calmodulin inhibitor W-7. The progressive increase in E with 30 pm-W-7 was largely reduced by dantrolene or high Mg2+ or BDM.4. In the presence of either dantrolene or BDM to prevent the effect of W-7 on SR Ca2+ release, exposure of the muscle to W-7 acutely suppressed about 3 % of E. This and the above results confirm that the plasmalemmal, calmodulin-dependent Ca2+-ATPase, although a qualitatively essential part of the Ca2+ homeostatic system of the cell, can only be responsible for a very minor part of the energy expenditure devoted to the homeostasis of Ca 2+. Active Ca2+ uptake by SR which, at least in the submicromolar range of Cai , is expected to be responsible for most of this Ca2+-dependent energy expenditure, might dissipate up to 25-40% of total metabolic energy in the intact mouse soleus under basal and near-basal conditions. MS 9801 22PHY 455

SUMMARY1. The steady-state heat production rate (1) of soleus muscles obtained from adult mice in various thyroid states was measured in a perfused microcalorimeter. The ouabain-suppressible fractions of E and 42K influx were compared and the energetic efficiency of active Na-K transport assessed.2. Hypothyroidism with plasma thyroxine concentrations below 1 jug/100 ml. was induced by pretreatment with 131I or perchlorate. In soleus muscles isolated from treated animals, mean E9 values were 2541 + 0 7 and 24-2 + 0 5 mcal.g wet wt.-'. min-for the 1311 and the perchlorate series respectively, i.e. about 30 % lower than the control level (36.3 + 1t5 mcal.g wetwt.-l.min-1). Followingtriiodothyronine treatment, X was increased by about 45 %.3. In muscles from hypothyroid (1311 and perchlorate series), euthyroid and hyperthyroid mice ouabain (10-3 M) induced a rapid decrease in ER of 1-6 + 0.1 and 1-4 + 0*1 2-5 + 0-2, and 4-3 + 0-6 mcal.g wet wt.-'.min-' respectively, i.e. between 6 and 8 % of1E.4. In muscles obtained from hypothyroid, euthyroid and hyperthyroid mice, the ouabain-suppressible component of 42K influx was 0-17 + 0 04, 031 + 0-02 and 0-45 + 0-02 Itmole. g wet wt.-. min-respectively. Whereas the total number of ouabain binding sites varied appreciably with the thyroid status, the Na-K contents of soleus or diaphragm muscles showed no significant changes.5. Notwithstanding the parallelism between the changes in basal X and ouabainsensitive components of E and K influx with the thyroid status, it is concluded that active Na-K transport cannot be considered a primary effector of thyroid thermogenesis in intact mammalian skeletal muscle. The direct contribution of active Na-K transport to this thermogenesis was indeed small compared with the over-all cellular energy dissipation.6. The minimum over-all energetic efficiency of the transport process in the intact muscle (30-35 %) was not dependent on the thyroid status.

Defects of both diet-induced thermogenesis and cold tolerance have been reported for streptozotocin-diabetic rats. Since brown adipose tissue (BAT) is a major effector of both diet- and cold-induced thermogenesis in the rat, the possible cause of these defects was investigated by comparing BAT metabolism under basal conditions and during activation by nerve stimulation, norepinephrine (NE), or octanoate addition in both streptozotocin-diabetic rats and in controls. The following metabolic indices were measured in rat interscapular BAT (IBAT): 1) tissue composition, 2) heat production rate as measured by direct microcalorimetry, 3) redox state of flavoproteins linked to the acyl-coenzyme A dehydrogenase pathway as measured by reflection spectrometry, 4) redox state of NAD(P) as measured by surface-emitted fluorescence, and 5) fatty acid activation and beta-oxidation activities in IBAT homogenate. In streptozotocin-diabetic rats, IBAT was atrophied (DNA content unmodified, protein and lipid content decreased). The basal and NE-stimulated total heat production rates showed a 75% and 56% decrease, respectively. The specific activity of fatty acid beta-oxidation as measured by flavoprotein redox state or enzymatically was decreased by 52% and 59%, respectively. The basal redox level of NAD(P) was about 3 times higher than in the controls and NE stimulation resulted in oxidation in contrast to the reduction observed in control tissues. These results show that the metabolic capacity of IBAT from streptozotocin-diabetic rats is decreased and further suggest that the reduced capacity for beta-oxidation contributes significantly to the metabolic alteration.