Interscapular Brown Adipose Tissue Mitochondria Research Articles

Metabolic clearance of oxaloacetate and mitochondrial complex II respiration: Divergent control in skeletal muscle and brown adipose tissue

At low inner mitochondrial membrane potential (ΔΨ) oxaloacetate (OAA) accumulates in the organelles concurrently with decreased complex II-energized respiration. This is consistent with ΔΨ-dependent OAA inhibition of succinate dehydrogenase. To assess the metabolic importance of this process, we tested the hypothesis that perturbing metabolic clearance of OAA in complex II-energized mitochondria would alter O2 flux and, further, that this would occur in both ΔΨ and tissue-dependent fashion. We carried out respiratory and metabolite studies in skeletal muscle and interscapular brown adipose tissue (IBAT) directed at the effect of OAA transamination to aspartate (catalyzed by the mitochondrial form of glutamic-oxaloacetic transaminase, Got2) on complex II-energized respiration. Addition of low amounts of glutamate to succinate-energized mitochondria at low ΔΨ increased complex II (succinate)-energized respiration in muscle but had little effect in IBAT mitochondria. The transaminase inhibitor, aminooxyacetic acid, increased OAA concentrations and impaired succinate-energized respiration in muscle but not IBAT mitochondria at low but not high ΔΨ. Immunoblotting revealed that Got2 expression was far greater in muscle than IBAT mitochondria. Because we incidentally observed metabolism of OAA to pyruvate in IBAT mitochondria, more so than in muscle mitochondria, we also examined the expression of mitochondrial oxaloacetate decarboxylase (ODX). ODX was detected only in IBAT mitochondria. In summary, at low but not high ΔΨ, mitochondrial transamination clears OAA preventing loss of complex II respiration: a process far more active in muscle than IBAT mitochondria. We also provide evidence that OAA decarboxylation clears OAA to pyruvate in IBAT mitochondria.

Membrane potential-dependent regulation of mitochondrial complex II by oxaloacetate in interscapular brown adipose tissue.

Classically, mitochondrial respiration responds to decreased membrane potential (ΔΨ) by increasing respiration. However, we found that for succinate‐energized complex II respiration in skeletal muscle mitochondria (unencumbered by rotenone), low ΔΨ impairs respiration by a mechanism culminating in oxaloacetate (OAA) inhibition of succinate dehydrogenase (SDH). Here, we investigated whether this phenomenon extends to far different mitochondria of a tissue wherein ΔΨ is intrinsically low, i.e., interscapular brown adipose tissue (IBAT). Also, to advance our knowledge of the mechanism, we performed isotopomer studies of metabolite flux not done in our previous muscle studies. In additional novel work, we addressed possible ways ADP might affect the mechanism in IBAT mitochondria. UCP1 activity, and consequently ΔΨ, were perturbed both by GDP, a well‐recognized potent inhibitor of UCP1 and by the chemical uncoupler carbonyl cyanide m‐chlorophenyl hydrazone (FCCP). In succinate‐energized mitochondria, GDP increased ΔΨ but also increased rather than decreased (as classically predicted under low ΔΨ) O2 flux. In GDP‐treated mitochondria, FCCP reduced potential but also decreased respiration. Metabolite studies by NMR and flux analyses by LC‐MS support a mechanism, wherein ΔΨ effects on the production of reactive oxygen alters the NADH/NAD+ ratio affecting OAA accumulation and, hence, OAA inhibition of SDH. We also found that ADP‐altered complex II respiration in complex fashion probably involving decreased ΔΨ due to ATP synthesis, a GDP‐like nucleotide inhibition of UCP1, and allosteric enzyme action. In summary, complex II respiration in IBAT mitochondria is regulated by UCP1‐dependent ΔΨ altering substrate flow through OAA and OAA inhibition of SDH.

Nitric oxide regulates mitochondrial re‐modelling in interscapular brown adipose tissue: ultrastructural and morphometric‐stereologic studies

As a complex, cell-specific process that includes both division and clear functional differentiation of mitochondria, mitochondriogenesis is regulated by numerous endocrine and autocrine factors. In the present ultrastructural study, in vivo effects of L-arginine-nitric oxide (NO)-producing pathway on mitochondriogenesis in interscapular brown adipose tissue (IBAT) were examined. For that purpose, adult Mill Hill hybrid hooded rats were receiving L-arginine, a substrate of NO synthases (NOSs), or N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOSs, as drinking liquids for 45 days. All experimental groups were divided into two sub-groups - acclimated to room temperature and cold. IBAT mitochondria were analyzed by transmission electron microscopy and stereology. L-Arginine treatment acted increasing the number of mitochondrial profiles per cell profile, as well as volume fraction of mitochondria per cell volume in animals maintained at room temperature. Cold-induced enhancement of number of mitochondrial profiles per cell profile was additionally increased in L-arginine-treated rats. Ultrastructural examinations of L-arginine-treated cold-acclimated animals clearly demonstrated thermogenically active mitochondria (larger size, lamellar, more numerous and well-ordered cristae in their profiles), which however were inactive in L-arginine-receiving animals kept at room temperature (small mitochondria, tubular cristae). By contrast, L-NAME treatment of rats acclimated to room temperature induced mitochondrial alterations characterized by irregular shape, short disorganized cristae and megamitochondria formation. These results showed that NO is a necessary factor for mitochondrial biogenesis and that it acts intensifying this process, but NO alone is not a sufficient stimulus for in vivo induction of mitochondriogenesis in brown adipocytes.

Nonshivering thermogenesis during acute cold exposure in adult and aged C57BL/6J mice.

In C57BL/6J adult and aged mice, housed at room temperature (22.5 +/- 1 degrees C), we measured O2 consumption and CO2 production and calculated metabolic heat production under conditions of anesthesia and myorelaxation during acute cold stimulation when body temperature was lowered 7.5 degrees C below control level. An independent group of mice was subjected to a three hour partial physical restraint at 6 degrees C and concentration of uncoupling protein (thermogenin) was measured in interscapular brown adipose tissue mitochondria at different times after cold exposure. Heat production under anesthesia and myorelaxation was about 57-66% lower than in nonanesthetized conditions, but increased significantly during cold stimulation in both age groups. Under anesthesia and myorelaxation before and during cold stimulation aged mice produced about 20% more heat than adult mice. Because in these experiments all sources of facultative thermogenesis, except nonshivering, were suppressed by anesthesia and myorelaxation, and because brown adipose tissue is the major source of nonshivering thermoproduction, we concluded that aged mice housed at room temperature have an increased thermogenesis in brown adipose tissue. This conclusion was also supported by the finding that the concentration of uncoupling protein measured in the mitochondria of brown adipose tissue after single cold exposure was significantly higher in aged than in adult mice. Therefore, we propose that the lower, cold-induced, heat production typically observed in nonanesthetized aged mice may reflect reduced thermogenic capacity of skeletal muscles. While aged mice have less brown adipose tissue than adult animals, the remaining brown adipose tissue may compensate by increasing the concentration of uncoupling protein.

Interaction of adrenalectomy and fenfluramine treatment on body weight, food intake and brown adipose tissue

Three experiments have examined the interaction of adrenalectomy and fenfluramine on food intake, body weight and the binding of guanosine-5′ diphosphate (GDP) to interscapular brown adipose tissue (IBAT). In the first experiment, GDP-binding by IBAT mitochondria from adrenalectomized or sham-operated animals was measured for 3 hr after one of 3 doses of fenfluramine. Fenfluramine stimulated GDP-binding at lower doses in the adrenalectomized animals than in the controls. In the first chronic experiment, adrenalectomy prevented the restoration of normal food intake observed 8–10 days after the beginning of fenfluramine treatment. Adrenalectomy also increased weight loss and enhanced GDP binding to mitochondria from IBAT in rats treated with fenfluramine. In the second chronic experiment, the combination of fenfluramine and adrenalectomy led to a progressive weight loss, continuing hypophagia and stimulation of GDP-binding by IBAT, whereas rats treated with fenfluramine alone showed a recovery of food intake at a stabilized but lower body weight. These data are consistent with the hypothesis that adrenalectomy and fenfluramine disable two separate components of the food intake system and that when combined, produce a profound and persisting disturbance in energy or nutrient balance.

Adrenalectomy and response to corticosterone and MSH in the genetically obese yellow mouse

Animals with the viable yellow (Avy/a) gene and their corresponding lean control black mice (a/a) were adrenalectomized or sham adrenalectomized, and changes in body weight, body composition, corticosterone, and GDP-binding to mitochondria isolated from interscapular brown adipose tissue (IBAT) were measured. Adrenalectomy slowed the weight gain of both the yellow obese mice and the black lean mice, but the reduction was greater in the yellow mice. Food intake was significantly reduced in the yellow mice. Adrenalectomy in the yellow mouse was associated with an increase in lean mass and a significant decrease in weights of fat depots. Blood glucose concentrations of the adrenalectomized yellow mice were reduced to levels similar to those of lean mice, but insulin levels, although lower than sham-adrenalectomized yellow mice, remained significantly higher than in lean animals. GDP binding to IBAT mitochondria increased after adrenalectomy in both phenotypes to values that were similar. Corticosterone replacement in adrenalectomized yellow mice produced a dose-dependent increase in body weight that was associated with a decrease in muscle weight and an increase in adipose tissue weight. Both desacetyl-melanocyte-stimulating hormone (MSH) and alpha-MSH interacted with corticosterone to increase body weight gain of adrenalectomized yellow mice. Desacetyl-MSH was more effective than alpha-MSH on increasing adipose tissue and liver weights. The effects of desacetyl-MSH on food intake, weight gain, and tissue weights were independent of the adrenal gland or of corticosterone.

Benzodiazepine binding sites in rat interscapular brown adipose tissue: Effect of cold environment, denervation and endocrine ablations

3H-Flunitrazepam (FNZP) binding was examined in a crude membrane fraction obtained from rat interscapular brown adipose tissue (IBAT). A single population of binding sites was apparent with dissociation constant (K D) = 0.47 ± 0.04 um and maximal number of binding sites (B max) = 31 ± 5 pmol.mg prot −1. From the activity of several benzodiazepine (BZP) analogs to compete for the binding, the peripheral nature of FNZP binding was tentatively established. Similar BZP binding sites were detectable in isolated IBAT mitochondria. Exposure of rats to 4°C for 15 days decreased B max significantly without affecting K D. Cold-induced decrease in B max of BZP binding was prevented by surgical IBAT denervation. Denervation prevented or impaired the increased activity of the mitochondrial markers succinate dehydrogenase and malate dehydrogenase in IBAT of cold-exposed rats, but did not affect monoamine oxidase activity. Hypophysectomy of rats decreased significantly both K D and B max of IBAT BZP binding. Thyroidectomy, adrenalectomy or ovari ectomy did not affect IBAT BZP binding parameters. The BZP analogs diazepam, clonazepan and Ro 5–4864 decreased significantly quanosine 5' -diphosphate binding (GDP) in IBAT mitochondria while co-incubation of Ro 5–4964 or clonazepam with the peripheral type BZP antagonist PK 11195 did not modify BZP activity on GDP binding. Our results indicate that BZP binding in rat IBAT may belong to the peripheral type, is decreased by a cold environment through activation of peripheral sympathetic nerves and is affected by hypophysectomy. BZP and GDP binding in IBAT mitochondria seem not to be functionally related.

Great adaptability of brown adipose tissue mitochondria to extreme ambient temperatures in control and cold-acclimated gerbils as compared with mice

1. 1. The gerbil ( Gerbillus campestris) is a desert rodent able to tolerate high (38°C) and low (−20°C) ambient temperatures, probably due to both its low resting metabolic rate in hot environment and its high peak metabolic rate in cold. 2. 2. Measurement of mitochondrial state IV respiration and cytochrome-oxidase activity (COX) were made in interscapular brown adipose tissue (IBAT), liver and hind limb muscles of gerbils and mice of nearly equal body mass, acclimated for 4 weeks at cold ambient temperature (CA) or reared at thermoneutrality (TN). 3. 3. The most striking difference between these two animal species appears to be in IBAT mitochondria: in TN animals, the level of state IV respiration and COX activity was lower in gerbils than in mice, but the cold acclimation-induced increase in these parameters was greater in gerbils than in mice. 4. 4. Alternatively, in gerbils as in mice, cold acclimation induced a reduction in muscle mitochondrial COX activity. No important change due to cold acclimation was observed in liver mitochondria, either in gerbils or in mice. 5. 5. As compared with mice, the lower state IV respiration in IBAT mitochondria from TN gerbils may explain their low RMR, whereas the higher COX activity of IBAT mitochondria from CA gerbils may explain their higher PMR. 6. 6. As a result of this great adaptability of BAT mitochondria, the gerbil seemed to be able to live in a wide range of ambient temperatures in its natural habitat.

Effect of age and gene dosage on brown adipose tissue of Zucker obese fa/fa rats.

Experiments were performed to investigate the effect of age and genotype on brown adipose tissue thermogenesis in Zucker rats. Specific [3H]GDP binding to interscapular brown adipose tissue mitochondria ( IBATM ) was reduced in 14-day-old preobese fa/fa rats and remained lower after weaning. A gene-dosage effect of the recessive fa gene was observed in 8- to 10-wk-old rats in both IBATM [3H]GDP binding and the thermic effect of a balanced meal (50 kJ Complan ), measured by indirect calorimetry. In each case the heterozygote (Fa/fa) group had a value intermediate between those of obese (fa/fa) and the homozygous lean (Fa/Fa) groups. Norepinephrine increased IBATM [3H]GDP binding to similar levels in lean (Fa/fa) and obese (fa/fa) rats and induced similar increases in oxygen consumption in Fa/Fa, Fa/fa, and fa/fa rats. It is concluded that the impaired, diet-related brown adipose tissue thermogenesis is closely related to the primary gene defect in the obese rat. This defect may result from misregulation of the autonomic nervous system.