Francisco J. García-Palmer

Cytochrome Oxidase Activity and Mitochondrial Gene Expression in Skeletal Muscle of Patients with Chronic Obstructive Pulmonary Disease

Several recent studies have suggested that skeletal muscle bioenergetics are abnormal in patients with chronic obstructive pulmonary disease (COPD). This study investigates the activity of cytochrome oxidase (COX), the terminal enzyme in the mitochondrial electron transport chain, and the expression of two mitochondrial DNA genes related to COX (mRNA of subunit I of COX [COX-I] and the RNA component of the 12S ribosomal subunit [12S rRNA]), in quadriceps femoris muscle biopsies obtained from COPD patients with various degrees of arterial hypoxemia, and from healthy sedentary control subjects of similar age. The activity of COX was measured spectrophotometrically in fresh tissue at 37 degrees C with excess substrate. RNA transcripts were measured using reverse transcription and polymerase chain reaction. The measurements of mRNA COX-I and 12S rRNA were normalized to the mRNA of actin, which is a housekeeping gene not influenced by hypoxia. We found that, compared with control subjects, COPD patients with chronic respiratory failure (PaO2 < 60 mm Hg) showed increased COX activity (p < 0.05). Further, the activity of COX was inversely related to arterial PO2 value (Rho -0.59, p < 0.01). The COX-I mRNA content was not different between patients and control subjects but patients with chronic respiratory failure had higher levels of 12S rRNA (p < 0.05), which were again inversely related to PaO2 (Rho -0.49, p < 0.05). These results indicate that the activity of COX is increased in skeletal muscle of patients with COPD and chronic respiratory failure, and they suggest that this is likely regulated at the translational level by increasing the number of mitochondrial ribosomes.

Skeletal Muscle of Female Rats Exhibit Higher Mitochondrial Mass and Oxidative- Phosphorylative Capacities Compared to Males

The effect of gender and caloric restriction on mitochondrial content and oxidative-phosphorylative capacities has been investigated in rat gastrocnemius muscle. Muscle protein, mitochondrial protein and DNA contents, enzymatic activities of mitochondrial oxidative and phosphorylative system, mitochondrial antioxidant enzymes, protein levels of complex IV (subunit I and IV) and ATPase, and the gene and protein expression of mitochondrial transcription factor A (TFAM), involved in mitochondrial replication and transcription, were measured in rats of both genders fed ad libitum and subjected to three months of 40% caloric restriction. Compared to males, gastrocnemius muscle of female rats showed higher mitochondrial DNA and protein contents, TFAM protein level, oxidative and phosphorylative machinery and activities, and glutathione peroxidase activity. In conclusion, the present data show a clear gender dimorphism in rat muscle mitochondrial features, which could explain the higher facility of females to adapt to altered metabolic energy situations.

Caloric Restriction Retards the Age-Related Decline in Mitochondrial Function of Brown Adipose Tissue

Caloric restriction (CR) has been shown to prevent the age-associated loss of mitochondrial function and biogenesis in several tissues such as liver, heart, and skeletal muscle. However, little is known about the effects of CR on a tissue in which the mitochondria have no adenosine triphosphate (ATP)-producing purpose but show a high degree of uncoupling, namely brown adipose tissue (BAT). Hence, the aim of the present study was to analyze the effect of long-term CR on BAT mitochondrial function and biogenesis. BAT mitochondria obtained from 24-month-old male and female rats previously subjected to 40% CR for 12 months were compared with mitochondria from old (24 months) and young (6 months) ad libitum fed rats. Old restricted rats compared to old ad libitum fed ones showed a reduction in BAT size with respect to fat content and adipocyte number. Mitochondrial DNA content in BAT increased with age and even more so in restricted rats, indicating a summative effect of age and CR on mitochondrial proliferation. CR induced resistance to lose total and mitochondrial protein, COX activity, and uncoupling capacity with advancing age, in relation with a lower decrease of mitochondrial transcription factor A (TFAM). In summary, our results demonstrate CR prevents the age-associated decline in mitochondrial function in BAT, probably in relation with a lower impairment of mitochondrial biogenesis.

Sex Differences in Brown Adipose Tissue Thermogenic Features During Caloric Restriction

Caloric restriction (CR) studies have shown that females rats conserve energy more efficiently, showing a higher resistance to weight loss and higher protection of vital organs mass than male rats. Gender-dependent inactivation of thermogenesis in brown adipose tissue (BAT) has been proposed as one of these possible energy conserving mechanisms. To study the changes underlying this inactivation in rats, a three month study with 40% CR was undertaken to unravel the effects on BAT recruitment. Under ad libitum conditions female rats had greater BAT recruitment and greater oxygen consumption than their male counterparts. Total and mitochondrial protein, as well as triglyceride and DNA content were more reduced in restricted female rats than in restricted males. Similarly, the levels of key BAT functional proteins (UCP1, LPL, HSL, TFAM) were more reduced in restricted females, whereas no changes were found in mitochondrial DNA levels (mtDNA) and OXPHOS activities in males and females. Furthermore, α 2A/β 3 adrenergic receptor ratio remained constant in male rats whereas in female rats CR increased 60%. In conclusion, our results suggest that female rats, whose BAT thermogenic activity is higher in ad libitum conditions, is depressed during CR. This inactivation involves the mitochondrial differentiation process and lipolytic system and could be due, at least in part, to the unfavourable adrenergic receptor balance for thermogenic activation.

Estradiol stimulates mitochondrial biogenesis and adiponectin expression in skeletal muscle.

Sexual dimorphism has been found in mitochondrial features of skeletal muscle, with female rats showing greater mitochondrial mass and function compared with males. Adiponectin is an insulin-sensitizing adipokine whose expression has been related to mitochondrial function and that is also expressed in skeletal muscle, where it exerts local metabolic effects. The aim of this research was to elucidate the role of sex hormones in modulation of mitochondrial function, as well as its relationship with adiponectin production in rat skeletal muscle. An in vivo study with ovariectomized Wistar rats receiving or not receiving 17β-estradiol (E2) (10 μg/kg per 48 h for 4 weeks) was carried out, in parallel with an assay of cultured myotubes (L6E9) treated with E2 (10 nM), progesterone (Pg; 1 μM), or testosterone (1 μM). E2 upregulated the markers of mitochondrial biogenesis and dynamics, and also of mitochondrial function in skeletal muscle and L6E9. Although in vivo E2 supplementation only partially restored the decreased adiponectin expression levels induced by ovariectomy, these were enhanced by E2 and Pg treatment in cultured myotubes, whereas testosterone showed no effects. Adiponectin receptor 1 expression was increased by E2 treatment, both in vivo and in vitro, but testosterone decreased it. In conclusion, our results are in agreement with the sexual dimorphism previously reported in skeletal muscle mitochondrial function and indicate E2 to be its main effector, as it enhances mitochondrial function and diminishes oxidative stress. Moreover, our data support the idea of the existence of a link between mitochondrial function and adiponectin expression in skeletal muscle, which could be modulated by sex hormones.

Skeletal muscle changes in patients with obstructive sleep apnoea syndrome

Previous studies have shown that chronic hypoxia leads to changes in skeletal muscle structure (fibre size and type) and activities of several bioenergetic enzymes. Whether this occurs also in conditions characterised by intermittent hypoxia, such as the obstructive sleep apnoea syndrome (OSAS), is unknown. To explore this possibility, we obtained a needle biopsy of the quadriceps femoris in 12 consecutive stable outpatients with severe OSAS (52 +/- 9 year, apnoea-hypopnoea index 70 +/- 14 h(-1)) (x +/- SD) and in six healthy volunteers (49 +/- 8 year), where we quantified fibre type, size and protein content, as well as phosphofructo-kinase (PFK) and cytochrome oxidase (CytOx) activities. We found that fibre-type distribution was similar in patients and controls. In contrast, the diameter of type II fibres (74 +/- 10 microm vs. 56 +/- 11 microm, P < 0.05) and protein content (100 +/- 14 vs. 88 +/- 8 microg/mg) was higher in patients with OSAS. Likewise, we observed upregulation of CytOx (0.93 +/- 0.38 vs. 0.40 +/- 0.22 microkat/mg protein, P < 0.01) and PFK activities (5.35 +/- 4.8 vs. 1.3 +/- 1.3 microkat/ mg protein, P < 0.05) in patients with OSAS. These results show that, paralleling which occurs in conditions characterised by continuous hypoxia, patients with OSAS (and intermittent hypoxia) also show structural and bioenergetic changes in their skeletal muscle.

Opposite effects of 17-β estradiol and testosterone on mitochondrial biogenesis and adiponectin synthesis in white adipocytes

Sexual dimorphism has been found in both mitochondrial functionality and adiponectin expression in white adipose tissue, with female rats presenting more functional mitochondria than males and greater adiponectin expression. However, little is known about the role of sex hormones in this dimorphism. The aim was to elucidate the role of sex hormones in mitochondrial biogenesis and dynamics and in adiponectin synthesis in white adipocytes, and also to provide new evidence of the link between these processes. 3T3-L1 preadipocytes were differentiated and treated either with 17-β estradiol (E₂; 10  nM), progesterone (Pg), testosterone (1  μM both), or a combination of Pg or testosterone with flutamide (FLT; 10  μM) or E₂ (1  μM). The markers of mitochondrial biogenesis and dynamics and adiponectin expression were analyzed. E₂ induced mitochondrial proliferation and differentiation in 3T3-L1, although testosterone showed opposite effects. Pg treatment stimulated proliferation but impaired differentiation. In concerns mitochondrial dynamics, these hormones promoted fusion over fission. FLT treatment indicated that Pg elicits its effects on mitochondrial dynamics through the androgen receptor. E₂ coadministration with testosterone or Pg reversed its effects. In conclusion, our results show that E₂ induces stimulation of mitochondrial biogenesis in white adipocytes in vitro, especially in situations that imply an impairment of mitochondrial function, whereas testosterone would have opposite effects. Moreover, testosterone and Pg alter mitochondrial dynamics by promoting fusion over fission, while E₂ stimulates both processes. All these alterations run in parallel with changes in adiponectin expression, thus suggesting the existence of a link between mitochondrial biogenesis and dynamics and adiponectin synthesis in white adipocytes.

The Serum Levels of 17β-estradiol, Progesterone and Triiodothyronine Correlate with Brown Adipose Tissue Thermogenic Parameters During Aging

Sex and thyroid hormones are among the factors modulating energy metabolism through regulation of mitochondrial oxidative capacity. Brown adipose tissue (BAT) in old female rats has been shown to maintain, better than males, the ability to produce heat when exposed to cold. Considering the decline that takes place in gonadal and thyroid function during aging, the aim of this work was to test whether the age-related hormonal status may be a potential mediator of the gender differences in BAT decline. COX activity, UCP1, mitochondrial respiration rate, mitochondrial DNA (mtDNA), levels of mitochondrial transcription factor A (TFAM), as well as the serum levels of sex steroids (17β-estradiol, progesterone and testosterone) and thyroid hormones (T3 and T4) were measured in 6-, 18-, and 24-month (mo) old male and female rats kept at 22 °C. Six mo-old female rats showed higher thermogenic features than males. Male rats at 18-mo showed a decrease in uncoupling activity compared to females. Both genders showed marked signs of atrophy in BAT of 24-mo-old rats, characterized by a decrease in total DNA, mitochondrial protein, COX and UCP1, whereas mtDNA was found to increase. Sex steroid hormones were well correlated with BAT parameters when both genders were considered, however, T3 was the hormone with the strongest positive correlations in female rats. In conclusion, our findings provide evidence suggesting that T3 may be a potential mediator of the sexual dimorphism in the effect of aging on the functional decline of BAT.

Mitochondrial Transcription Factor A (TFAM) is Increased in Rat Embryo During Placentation and Associated with Mitochondrial Differentiation

In the current study, the mitochondrial proliferationdifferentiation process was investigated in rat embryo during the placentation process, straight after organogenesis, when there is an important oxidative metabolism activation. For this purpose, on gestational days 11, 12 and 13 we studied the mitochondrial DNA (mtDNA) content and the relative gene expression of proteins involved in mtDNA replication (mitochondrial single strand DNA binding protein (mtSSB)), mtDNA transcription (mitochondrial transcription factor A (TFAM)), as well as in mitochondrial function (cytochrome c oxidase subunit I (COXI)). The results indicated that during placentation important changes in mitochondrial proliferation-differentiation process take place in rat embryo. There is a great decrease in cellular mtDNA content and a rise in the ratio between TFAM and mtDNA accompanied by an increase in COXI gene expression. Thus, we can conclude that on gestational day 13 mitochondrial differentiation predominates over mitochondrial proliferation in embryo cells. Besides, our work reveals that in a physiological condition such as embryonic development the TFAM levels change in order to regulate the transcriptional activity of mtDNA.

Rat Brown Adipose Tissue Thermogenic Features are Altered During Mid-Pregnancy

Brown adipose tissue (BAT) thermogenesis is inhibited during late-pregnancy and lactation in the rat. However, scarce information concerning BAT functionality during mid-pregnancy is available. The aim of this work was to investigate uncoupling proteins and leptin expression during placentation in rat BAT as well as other key parameters in the thermogenic function of the tissue. BAT mitochondrial content was found to be reduced 50% in 11 and 13 day pregnant rats as compared to nonpregnant controls, although uncoupling protein 1 (UCP1) content was not modified. Furthermore, UCP3 mRNA levels were found to be highly increased during this period. β3-adrenergic receptor (β3-AR) decreased expression resulted in a higher α2/β3 ratio. Finally, leptin mRNA levels in BAT were found to be 3-fold up-regulated in pregnant animals. In conclusion, we show the existence of profound changes in thermogenic features in BAT during gestational days 11 and 13, pointing to the importance of this tissue during mid-pregnancy.