Maria Moreno

ACTION OF THYROID HORMONES AT THE CELLULAR LEVEL : THE MITOCHONDRIAL TARGET

Thyroid hormones exert profound effects on the energy metabolism. An inspection of the early and more recent literature shows that several targets at the cellular level have been identified. Since their effects on the nuclear signalling pathway have already been well‐defined and extensively reviewed, this article focuses on the regulation of mitochondrial activity by thyroid hormones. Mitochondria, by virtue of their biochemical functions, are a natural candidate as a direct target for the calorigenic effects of thyroid hormones. To judge from results coming from various laboratories, it is quite conceivable that mitochondrial activities are regulated both directly and indirectly. Not only triiodo‐L‐thyronine, but also diiodothyronines are active in regulating the energy metabolism. They influence the resting metabolism in rats with 3,5‐diiodo‐L‐thyronine seeming to show a clearer effect.

Oxidative stress and vascular remodelling

Oxidative stress plays an important role in the pathophysiology of vascular diseases. Reactive oxygen species, especially superoxide anion and hydrogen peroxide, are important signalling molecules in cardiovascular cells. Enhanced superoxide production increases nitric oxide inactivation and leads to an accumulation of peroxynitrites and hydrogen peroxide. Reactive oxygen species participate in growth, apoptosis and migration of vascular smooth muscle cells, in the modulation of endothelial function, including endothelium‐dependent relaxation and expression of proinflammatory phenotype, and in the modification of the extracellular matrix. All these events play important roles in vascular diseases such as hypertension, suggesting that the sources of reactive oxygen species and the signalling pathways that they modify may represent important therapeutic targets. Potential sources of vascular superoxide production include NADPH‐dependent oxidases, xanthine oxidases, lipoxygenases, mitochondrial oxidases and nitric oxide synthases. Studies performed during the last decade have shown that NADPH oxidase is the most important source of superoxide anion in phagocytic and vascular cells. Evidence from experimental animal and human studies suggests a significant role of NADPH oxidase activation in the vascular remodelling and endothelial dysfunction found in cardiovascular diseases.

Thyroid hormone and uncoupling proteins

Thyroid hormone (TH/T3) exerts many of its effects on energy metabolism by affecting gene transcription. However, although this is an important target for T3, only a limited number of T3‐responsive genes have been identified and studied. Among these, the genes for uncoupling proteins (UCPs) have attracted the interest of scientists. Although the role of UCP1 seems quite well established, uncertainty surrounds the physiological function of the recently discovered UCP1 analogs, UCP2 and UCP3. The literature suggests that T3 affects both the expression and the activity of each of these UCPs but further studies are needed to establish whether the mechanisms activated by the hormone are the same. Recently, because of their larger range of expression, much attention has been devoted to UCP2 and UCP3. Most detailed studies on the involvement of these proteins as mediators of the effects of T3 on metabolism have focused on UCP3 because of its expression in skeletal muscle. T3 seems to be unique in having the ability to stimulate the expression and activity of UCP3 and this may be related to the capacity of T3 to activate the integrated biochemical processes linked to UCP activity, such as those related to fatty acids, coenzyme Q and free radicals.

Fuel economy in food-deprived skeletal muscle: signaling pathways and regulatory mechanisms

Energy deprivation poses a tremendous challenge to skeletal muscle. Glucose (ATP) depletion causes muscle fibers to undergo rapid adaptive changes toward the use of fatty acids (instead of glucose) as fuel. Physiological situations involving energy deprivation in skeletal muscle include exercise and fasting. A vast body of evidence is available on the signaling pathways that lead to structural/metabolic changes in muscle during exercise and endurance training. In contrast, only recently has a systematic, overall picture been obtained of the signaling processes (and their kinetics and sequential order) that lead to adaptations of the muscle to the fasting state. It has become clear that the reaction of the organism to food restraint or deprivation involves a rapid signaling process causing skeletal muscles, which generally use glucose as their predominant fuel, to switch to the use of fat as fuel. Efficient sensing of glucose depletion in skeletal muscle guarantees maintained activity in those tissues that rely entirely on glucose (such as the brain). To metabolize fatty acids, skeletal muscle needs to activate complex transcription, translation, and phosphorylation pathways. Only recently has it become clear that these pathways are interrelated and tightly regulated in a rapid, transient manner. Food deprivation may trigger these responses with a timing/intensity that differs among animal species and that may depend on their individual ability to induce structural/metabolic changes that serve to safeguard whole‐body energy homeostasis in the longer term. The increased cellular AMP/ATP ratio induced by food deprivation, which results in activation of AMP‐activated protein kinase (AMPK), initiates a rapid signaling process, resulting in the recruitment of factors mediating the structural/ metabolic shift in skeletal muscle toward this change in fuel usage. These factors include peroxisome prolifera‐tor‐activated receptor (PPAR)γ coactivator‐1α (PGC‐1α), PPARδ, and their target genes, which are involved in the formation of oxidative muscle fibers, mitochon‐drial biogenesis, oxidative phosphorylation, and fatty acid oxidation. Fatty acids, besides being the fuel for mitochondrial oxidation, have been identified as important signaling molecules regulating the transcription and/or activity of the genes or gene products involved in fatty acid metabolism during food deprivation. It is thus becoming increasingly clear that fatty acids determine the economy of their own usage. We discuss the order of events from the onset of food deprivation and their importance.—de Lange P., Moreno, M., Silvestri, E., Lombardi, A., Goglia, F., Lanni A. Fuel economy in food‐deprived skeletal muscle: signaling pathways and regulatory mechanisms. FASEB J. 21, 3431–3441 (2007)

Expression of uncoupling protein‐3 and mitochondrial activity in the transition from hypothyroid to hyperthyroid state in rat skeletal muscle

We sought a correlation between rat skeletal muscle triiodothyronine (T3)‐mediated regulation of uncoupling protein‐3 (UCP3) expression and mitochondrial activity. UCP3 mRNA expression increased strongly during the hypothyroid‐hyperthyroid transition. The rank order of mitochondrial State 3 and State 4 respiration rates was hypothyroid

3,5-Diiodo-L-thyronine powerfully reduces adiposity in rats by increasing the burning of fats

The effect of thyroid hormones on metabolism has long supported their potential as drugs to stimulate fat reduction, but the concomitant induction of a thyrotoxic state has greatly limited their use. Recent evidence suggests that 3,5‐diiodo‐L‐thyronine (T2), a naturally occurring iodothyronine, stimulates metabolic rate via mechanisms involving the mitochondrial apparatus. We examined whether this effect would result in reduced energy storage. Here, we show that T2 administration to rats receiving a high‐fat diet (HFD) reduces both adiposity and body weight gain without inducing thyrotoxicity. Rats receiving HFD + T2 showed (when compared with rats receiving HFD alone) a 13% lower body weight, a 42% higher liver fatty acid oxidation rate, ∼50% less fat mass, a complete disappearance of fat from the liver, and significant reductions in the serum triglyceride and cholesterol levels (−52% and −18%, respectively). Thyroid hormones and thyroid‐stimulating hormone (TSH) serum levels were not influenced by T2 administration. The biochemical mechanism underlying the effects of T2 on liver metabolism involves the carnitine palmitoyl‐transferase system and mitochondrial uncoupling. If the results hold true for humans, pharmacological administration of T2 might serve to counteract the problems associated with overweight, such as accumulation of lipids in liver and serum, without inducing thyrotoxicity. However, the results reported here do not exclude deleterious effects of T2 on a longer time scale as well as do not show that T2 acts in the same way in humans.

Gene expression profiling in whole blood of patients with coronary artery disease.

Owing to the dynamic nature of the transcriptome, gene expression profiling is a promising tool for discovery of disease-related genes and biological pathways. In the present study, we examined gene expression in whole blood of 12 patients with CAD (coronary artery disease) and 12 healthy control subjects. Furthermore, ten patients with CAD underwent whole-blood gene expression analysis before and after the completion of a cardiac rehabilitation programme following surgical coronary revascularization. mRNA and miRNA (microRNA) were isolated for expression profiling. Gene expression analysis identified 365 differentially expressed genes in patients with CAD compared with healthy controls (175 up- and 190 down-regulated in CAD), and 645 in CAD rehabilitation patients (196 up- and 449 down-regulated post-rehabilitation). Biological pathway analysis identified a number of canonical pathways, including oxidative phosphorylation and mitochondrial function, as being significantly and consistently modulated across the groups. Analysis of miRNA expression revealed a number of differentially expressed miRNAs, including hsa-miR-140-3p (control compared with CAD, P=0.017), hsa-miR-182 (control compared with CAD, P=0.093), hsa-miR-92a and hsa-miR-92b (post- compared with pre-exercise, P<0.01). Global analysis of predicted miRNA targets found significantly reduced expression of genes with target regions compared with those without: hsa-miR-140-3p (P=0.002), hsa-miR-182 (P=0.001), hsa-miR-92a and hsa-miR-92b (P=2.2×10−16). In conclusion, using whole blood as a ‘surrogate tissue’ in patients with CAD, we have identified differentially expressed miRNAs, differentially regulated genes and modulated pathways which warrant further investigation in the setting of cardiovascular function. This approach may represent a novel non-invasive strategy to unravel potentially modifiable pathways and possible therapeutic targets in cardiovascular disease.

Association of increased phagocytic NADPH oxidase-dependent superoxide production with diminished nitric oxide generation in essential hypertension

Objective Oxidative stress has been implicated in the pathogenesis of hypertension and its complications through alterations in nitric oxide (NO) metabolism. This study was designed to investigate whether a relationship exists between phagocytic nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent superoxide anion (•O2−) production and NO generation in patients with essential hypertension. Methods Superoxide production was assayed by chemiluminescence under baseline and stimulated conditions on mononuclear cells obtained from hypertensives (n = 51) and normotensives (n = 43). NO production was evaluated by determining serum NO metabolites, nitrate plus nitrite (NOx). Results Although there were no differences in baseline •O2− production between normotensives and hypertensives, the •O2− production in phorbol myristate acetate (PMA)-stimulated mononuclear cells was increased (P < 0.05) in hypertensives compared with normotensives. The PMA-induced •O2− production was completely abolished by apocynin, a specific inhibitor of NADPH oxidase. Moreover, stimulation of •O2− production by angiotensin II and endothelin-1 was higher (P < 0.05) in cells from hypertensives than in cells from normotensives. In addition, diminished (P < 0.001) serum NOx was detected in hypertensives compared with normotensives. Interestingly, an inverse correlation (r = 0.493, P < 0.01) was found between •O2− production and NOx in hypertensives. Conclusions Generation of •O2− mainly dependent on NADPH oxidase is abnormally enhanced in stimulated mononuclear cells from hypertensives. It is suggested that this alteration could be involved in the diminished NO production observed in these patients.

Preliminary characterisation of the promoter of the human p22phox gene: identification of a new polymorphism associated with hypertension

The p22phox subunit is an essential protein in the activation of NAD(P)H oxidase. Here we report the preliminary characterisation of the human p22phox gene promoter. The p22phox promoter contains TATA and CCAC boxes and Sp1, γ‐interferon and nuclear factor κB sites. We screened for mutations in the p22phox promoter and identified a new polymorphism, localised at position −930 from the ATG codon, which was associated with hypertension. Mutagenesis experiments showed that the G allele had higher promoter activity than the A allele. These results suggest that the −930A/G polymorphism in the p22phox promoter may be a novel genetic marker associated with hypertension.

Functional Effect of the p22phox −930A/G Polymorphism on p22phox Expression and NADPH Oxidase Activity in Hypertension

Oxidative stress induced by superoxide is implicated in hypertension. NADPH oxidase is the main source of superoxide in phagocytic and vascular cells, and the p22phox subunit is involved in NADPH oxidase activation. Recently we reported an association of −930A/G polymorphism in the human p22phox gene promoter with hypertension. This study was designed to investigate the functional role of this polymorphism in hypertension. We thus investigated the relationships between the −930A/G polymorphism and p22phox expression and NADPH oxidase–mediated superoxide production in phagocytic cells from 70 patients with essential hypertension and 70 normotensive controls. Genotyping of the polymorphism was performed by restriction fragment length polymorphism. NADPH oxidase activity was determined by chemiluminescence assays, and p22phox mRNA and protein expression was measured by Northern and Western blotting, respectively. Compared with hypertensive subjects with the AA/AG genotype, hypertensive subjects with the GG genotype exhibited increased (P <0.05) phagocytic p22phox mRNA (1.26±0.06 arbitrary unit [AU] versus 0.99±0.03 AU) and protein levels (0.58±0.05 AU versus 0.34±0.04 AU) and enhanced NADPH oxidase activity (1998±181 counts/s versus 1322±112 counts/s). No differences in these parameters were observed among genotypes in normotensive cells. Transfection experiments on vascular smooth muscle cells showed that the A-to-G substitution of this polymorphism produced an increased reporter gene expression in hypertensive cells. Nitric oxide production, as assessed by measurement of serum nitric oxide metabolites, was lower in GG hypertensive subjects than in AA/AG hypertensive subjects. In conclusion, these results suggest that hypertensive subjects carrying the GG genotype of the p22phox −930A/G polymorphism are highly exposed to NADPH oxidase-mediated oxidative stress.