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1.
J Biol Chem ; 292(34): 14176-14187, 2017 08 25.
Article in English | MEDLINE | ID: mdl-28663370

ABSTRACT

Mitochondrial oxidation of nutrients is tightly regulated in response to the cellular environment and changes in energy demands. In vitro studies evaluating the mitochondrial capacity of oxidizing different substrates are important for understanding metabolic shifts in physiological adaptations and pathological conditions, but may be influenced by the nutrients present in the culture medium or by the utilization of endogenous stores. One such influence is exemplified by the Crabtree effect (the glucose-mediated inhibition of mitochondrial respiration) as most in vitro experiments are performed in glucose-containing media. Here, using high-resolution respirometry, we evaluated the oxidation of endogenous or exogenous substrates by cell lines harboring different metabolic profiles. We found that a 1-h deprivation of the main energetic nutrients is an appropriate strategy to abolish interference of endogenous or undesirable exogenous substrates with the cellular capacity of oxidizing specific substrates, namely glutamine, pyruvate, glucose, or palmitate, in mitochondria. This approach primed mitochondria to immediately increase their oxygen consumption after the addition of the exogenous nutrients. All starved cells could oxidize exogenous glutamine, whereas the capacity for oxidizing palmitate was limited to human hepatocarcinoma Huh7 cells and to C2C12 mouse myoblasts that differentiated into myotubes. In the presence of exogenous glucose, starvation decreased the Crabtree effect in Huh7 and C2C12 cells and abrogated it in mouse neuroblastoma N2A cells. Interestingly, the fact that the Crabtree effect was observed only for mitochondrial basal respiration but not for the maximum respiratory capacity suggests it is not caused by a direct effect on the electron transport system.


Subject(s)
Energy Metabolism , Gene Expression Regulation, Enzymologic , Mitochondria, Liver/metabolism , Mitochondria, Muscle/metabolism , Mitochondria/metabolism , Oxidative Stress , Animals , Cell Line , Cell Line, Tumor , Electron-Transferring Flavoproteins/metabolism , Glucose/metabolism , Glutamine/metabolism , Humans , Kinetics , Mice , Mitochondria/enzymology , Mitochondria, Liver/enzymology , Mitochondria, Muscle/enzymology , Organ Specificity , Oxidation-Reduction , Oxidative Phosphorylation , Palmitic Acid/metabolism , Pyruvic Acid/metabolism
2.
J. Biol. Chem. ; 292(34): 14176-14187, 2017.
Article in English | Sec. Est. Saúde SP, SESSP-IBPROD, Sec. Est. Saúde SP | ID: but-ib15054

ABSTRACT

Mitochondrial oxidation of nutrients is tightly regulated in response to the cellular environment and changes in energy demands. In vitro studies evaluating the mitochondrial capacity of oxidizing different substrates are important for understanding metabolic shifts in physiological adaptations and pathological conditions, but may be influenced by the nutrients present in the culture medium or by the utilization of endogenous stores. One such influence is exemplified by the Crabtree effect (the glucose-mediated inhibition of mitochondrial respiration) as most in vitro experiments are performed in glucose-containing media. Here, using high-resolution respirometry, we evaluated the oxidation of endogenous or exogenous substrates by cell lines harboring different metabolic profiles. We found that a 1-h deprivation of the main energetic nutrients is an appropriate strategy to abolish interference of endogenous or undesirable exogenous substrates with the cellular capacity of oxidizing specific substrates, namely glutamine, pyruvate, glucose, or palmitate, in mitochondria. This approach primed mitochondria to immediately increase their oxygen consumption after the addition of the exogenous nutrients. All starved cells could oxidize exogenous glutamine, whereas the capacity for oxidizing palmitate was limited to human hepatocarcinoma Huh7 cells and to C2C12 mouse myoblasts that differentiated into myotubes. In the presence of exogenous glucose, starvation decreased the Crabtree effect in Huh7 and C2C12 cells and abrogated it in mouse neuroblastoma N2A cells. Interestingly, the fact that the Crabtree effect was observed only for mitochondrial basal respiration but not for the maximum respiratory capacity suggests it is not caused by a direct effect on the electron transport system.

3.
Endocrinology ; 155(8): 2881-91, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24914935

ABSTRACT

Menopause is associated with increased visceral adiposity and disrupted glucose homeostasis, but the underlying molecular mechanisms related to these metabolic changes are still elusive. Brown adipose tissue (BAT) plays a key role in energy expenditure that may be regulated by sexual steroids, and alterations in glucose homeostasis could precede increased weight gain after ovariectomy. Thus, the aim of this work was to evaluate the metabolic pathways in both the BAT and the liver that may be disrupted early after ovariectomy. Ovariectomized (OVX) rats had increased food efficiency as early as 12 days after ovariectomy, which could not be explained by differences in feces content. Analysis of isolated BAT mitochondria function revealed no differences in citrate synthase activity, uncoupling protein 1 expression, oxygen consumption, ATP synthesis, or heat production in OVX rats. The addition of GDP and BSA to inhibit uncoupling protein 1 decreased oxygen consumption in BAT mitochondria equally in both groups. Liver analysis revealed increased triglyceride content accompanied by decreased levels of phosphorylated AMP-activated protein kinase and phosphorylated acetyl-CoA carboxylase in OVX animals. The elevated expression of gluconeogenic enzymes in OVX and OVX + estradiol rats was not associated with alterations in glucose tolerance test or in serum insulin but was coincident with higher glucose disposal during the pyruvate tolerance test. Although estradiol treatment prevented the ovariectomy-induced increase in body weight and hepatic triglyceride and cholesterol accumulation, it was not able to prevent increased gluconeogenesis. In conclusion, the disrupted liver glucose homeostasis after ovariectomy is neither caused by estradiol deficiency nor is related to increased body mass.


Subject(s)
Adipose Tissue, Brown/metabolism , Estradiol/physiology , Liver/metabolism , Menopause/metabolism , Weight Gain , Animals , Female , Glucose/metabolism , Obesity/etiology , Obesity/metabolism , Ovariectomy , Rats , Rats, Wistar , Thermogenesis , Triglycerides/metabolism
4.
Biosci Rep ; 32(1): 53-9, 2012 Feb.
Article in English | MEDLINE | ID: mdl-21561434

ABSTRACT

The UCP1 [first UCP (uncoupling protein)] that is found in the mitochondria of brown adipocytes [BAT (brown adipose tissue)] regulates the heat production, a process linked to non-shivering thermogenesis. The activity of UCP1 is modulated by GDP and fatty acids. In this report, we demonstrate that respiration and heat released by BAT mitochondria vary depending on the respiratory substrate utilized and the coupling state of the mitochondria. It has already been established that, in the presence of pyruvate/malate, BAT mitochondria are coupled by faf-BSA (fatty-acid-free BSA) and GDP, leading to an increase in ATP synthesis and mitochondrial membrane potential along with simultaneous decreases in both the rates of respiration and heat production. Oleate restores the uncoupled state, inhibiting ATP synthesis and increasing the rates of both respiration and heat production. We now show that in the presence of succinate: (i) the rates of uncoupled mitochondria respiration and heat production are five times slower than in the presence of pyruvate/malate; (ii) faf-BSA and GDP accelerate heat and respiration as a result and, in coupled mitochondria, these two rates are accelerated compared with pyruvate/malate; (iii) in spite of the differences in respiration and heat production noted with the two substrates, the membrane potential and the ATP synthesized were the same; and (iv) oleate promoted a decrease in heat production and respiration in coupled mitochondria, an effect different from that observed using pyruvate/malate. These effects are not related to the production of ROS (reactive oxygen species). We suggest that succinate could stimulate a new route to heat production in BAT mitochondria.


Subject(s)
Adipose Tissue, Brown/metabolism , Fatty Acids/metabolism , Guanosine Diphosphate/metabolism , Mitochondria/metabolism , Adenosine Triphosphate/biosynthesis , Animals , Cell Respiration/drug effects , Fatty Acids/pharmacology , Hydrogen Peroxide/metabolism , Malates/metabolism , Malates/pharmacology , Male , Oleic Acid/metabolism , Oleic Acid/pharmacology , Pyruvic Acid/metabolism , Pyruvic Acid/pharmacology , Rats , Rats, Wistar , Rotenone/pharmacology , Succinic Acid/metabolism , Succinic Acid/pharmacology , Thermogenesis/drug effects , Uncoupling Agents/pharmacology
5.
PLoS One ; 5(3): e9439, 2010 Mar 02.
Article in English | MEDLINE | ID: mdl-20209153

ABSTRACT

Brown adipose tissue (BAT) mitochondria thermogenesis is regulated by uncoupling protein 1 (UCP 1), GDP and fatty acids. In this report, we observed fusion of the endoplasmic reticulum (ER) membrane with the mitochondrial outer membrane of rats BAT. Ca(2+)-ATPase (SERCA 1) was identified by immunoelectron microscopy in both ER and mitochondria. This finding led us to test the Ca(2+) effect in BAT mitochondria thermogenesis. We found that Ca(2+) increased the rate of respiration and heat production measured with a microcalorimeter both in coupled and uncoupled mitochondria, but had no effect on the rate of ATP synthesis. The Ca(2+) concentration needed for half-maximal activation varied between 0.08 and 0.11 microM. The activation of respiration was less pronounced than that of heat production. Heat production and ATP synthesis were inhibited by rotenone and KCN. Liver mitochondria have no UCP1 and during respiration synthesize a large amount of ATP, produce little heat, GDP had no effect on mitochondria coupling, Ca(2+) strongly inhibited ATP synthesis and had little or no effect on the small amount of heat released. These finding indicate that Ca(2+) activation of thermogenesis may be a specific feature of BAT mitochondria not found in other mitochondria such as liver.


Subject(s)
Adipose Tissue, Brown/metabolism , Adipose Tissue/metabolism , Endoplasmic Reticulum/metabolism , Intracellular Membranes/metabolism , Mitochondria/metabolism , Adenosine Triphosphate/chemistry , Animals , Calorimetry/methods , Cyanates/chemistry , Male , Mitochondria, Liver/metabolism , Oxygen Consumption , Rats , Rats, Wistar , Rotenone/pharmacology
6.
Am J Physiol Heart Circ Physiol ; 297(2): H556-63, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19525379

ABSTRACT

Short-term response to cold promotes a small but significant rise in serum T3 in euthyroid rabbits, where the heart is an important target of T3 action. In this work, we measured changes in sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2a) and phospholamban (PLB) in hearts of hypo- and hyperthyroid rabbits and compared them with modifications induced by short- and long-term cold exposure. Short-term cold exposure promotes a small increase in T3 and, similar to hyperthyroidism, induces an increase of heart SERCA2a expression. The total PLB content does not change in hyperthyroidism, but short-term cold exposure promotes a significant decrease in total PLB and an increase in the ratio between phosphorylated and total PLB. The temperature of a given tissue depends on the balance between the heat provided by blood circulation and the rate of heat production by the tissue. In an attempt to evaluate the heat contribution of cardiac tissue, we measured mitochondrial respiration in permeabilized cardiac muscle and heat produced by cardiac sarcoplasmic reticulum (SR) during Ca(2+) transport. We observed that there was an increase in oxygen consumption and heat production during Ca(2+) transport by cardiac SR in both hyperthyroidism and short-term cold exposure. In contrast, both the mitochondrial respiration rate and heat derived from Ca(2+) transport were decreased in hypothyroid rabbits. The heart changes in oxygen consumption, SERCA2a-PLB ratio, and Ca(2+)-ATPase activity detected during short-term cold exposure were abolished after cold adaptation. We hypothesize that the transient rise in serum T3 contributes to the short-term response to cold exposure.


Subject(s)
Body Temperature Regulation/physiology , Calcium-Binding Proteins/metabolism , Myocardium/metabolism , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Triiodothyronine/metabolism , Adaptation, Physiological/physiology , Animals , Calcium/metabolism , Citrate (si)-Synthase/metabolism , Cold Temperature , Energy Metabolism/physiology , Hot Temperature , Hyperthyroidism/metabolism , Hypothyroidism/metabolism , Male , Mitochondria, Heart/metabolism , Oxygen Consumption/physiology , Rabbits , Thyroxine/metabolism , Thyroxine/pharmacology
7.
Endocrinology ; 149(12): 6262-71, 2008 Dec.
Article in English | MEDLINE | ID: mdl-18703625

ABSTRACT

Brown adipose tissue (BAT) is involved in rat and mice thermoregulation, and heat produced by BAT depends on the concerted action of thyroid hormones and catecholamines. Little is known about cold-induced thermogenesis in mammals that have little or no BAT, such as rabbits. In these animals, thermogenesis primarily occurs in skeletal muscle. In this work, we have studied the effect of cold acclimation (4 C for 10 d) in normal and hypothyroid rabbits. It is known that hypothyroid rats die after a few hours of cold exposure. We now show that, different from rats, hypothyroid rabbits sustain their body temperature and survive after 10 d cold exposure. When compared with rabbits kept at room temperature, the muscles of cold-exposed rabbits showed a dark red color characteristic of oxidative muscle fibers. According to this pattern, we observed that in both normal and hypothyroid rabbits, cold exposure promotes an increase in oxygen consumption by skeletal muscle mitochondria. Moreover, in red muscle, cold acclimation induces an increase in the expression and activity of sarcoplasmic reticulum Ca(2+) ATPase isoform 1 (SERCA1), one of the muscle enzymes involved in heat production. We conclude that rabbit cold tolerance is probably related to increased muscle oxidative metabolism and heat production by SERCA1 and that these changes are not completely dependent on normal thyroid function.


Subject(s)
Cold Temperature , Hypothyroidism/metabolism , Mitochondria, Muscle/metabolism , Muscle, Skeletal/metabolism , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Animals , Blotting, Western , Body Weight , Eating , Hypothyroidism/blood , Hypothyroidism/physiopathology , Male , Mitochondria, Muscle/physiology , Muscle, Skeletal/physiology , Oxygen Consumption/physiology , Rabbits , Thermogenesis , Thyroxine/blood , Time Factors , Triiodothyronine/blood
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