Fernando Goglia

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.

Fenofibrate prevents and reduces body weight gain and adiposity in diet-induced obese rats

Fibrates are hypolipidemic drugs that activate the peroxisome proliferator‐activated receptors. Since fibrates may also increase energy expenditure, we investigated whether fenofibrate (FF) had this effect in diet‐induced obese rats. A 2‐month administration of a high‐fat palatable diet to adult rats increased body weight by 25% and white adipose mass by 163% compared with a standard diet. These effects were prevented by FF, both when administered for the 2 months of high‐fat feeding and when given for only the second month. Consequently, FF‐treated rats had a final body weight and white adipose tissue mass similar to untreated animals on the standard diet. FF also increased resting metabolic rate, hepatic peroxisomal and mitochondrial palmitoyl‐dependent oxygen uptake and mRNA levels of acyl‐CoA oxidase and lipoprotein lipase. Finally, FF lowered mRNA levels of uncoupling protein‐2 and did not affect mitochondrial respiration in skeletal muscle. Therefore, FF seems to act as a weight‐stabilizer mainly through its effect on liver metabolism.

Defining the transcriptomic and proteomic profiles of rat ageing skeletal muscle by the use of a cDNA array, 2D- and Blue native-PAGE approach.

We defined the transcriptomic and proteomic profiles of rat ageing skeletal muscle using a combined cDNA array, 2D- and Blue native-PAGE approach. This was allowed to obtain an overview of the interrelated events leading to the transcriptome/proteome/mitoproteome changes likely to underlie the structural/metabolic features of aged skeletal muscle. The main differences were found in genes/proteins related to energy metabolism, mitochondrial pathways, myofibrillar filaments, and detoxification. Concerning the abundance of mitochondrial OXPHOS complexes as well as their supramolecular organization and activity, mitochondria from old rats, when compared with those from young rats, contained significantly lower amounts of complex I (NADH:ubiquinone oxidoreductase), V (FoF1-ATP synthase), and III (ubiquinol:cytochrome c oxidoreductase). The same mitochondria contained a significantly larger amount of complex II (succinate:ubiquinone oxidoreductase), but an unchanged amount of complex IV (cytochrome c oxidase, COX). When comparing the supercomplex profiles between young and old muscle mitochondria, the densitometric analysis revealed that lighter supercomplexes were significantly reduced in older mitochondria, and that in the older group the major supercomplex bands were those representing heavier supercomplexes, likely suggesting a compensatory mechanism that, in ageing muscle, is functionally directed towards substrate channeling and catalytic enhancement advantaging the respirosome.

3,5-Diiodo-l-thyronine rapidly enhances mitochondrial fatty acid oxidation rate and thermogenesis in rat skeletal muscle: AMP-activated protein kinase involvement

Triiodothyronine regulates energy metabolism and thermogenesis. Among triiodothyronine derivatives, 3,5-diiodo-l-thyronine (T(2)) has been shown to exert marked effects on energy metabolism by acting mainly at the mitochondrial level. Here we investigated the capacity of T(2) to affect both skeletal muscle mitochondrial substrate oxidation and thermogenesis within 1 h after its injection into hypothyroid rats. Administration of T(2) induced an increase in mitochondrial oxidation when palmitoyl-CoA (+104%), palmitoylcarnitine (+80%), or succinate (+30%) was used as substrate, but it had no effect when pyruvate was used. T(2) was able to 1) activate the AMPK-ACC-malonyl-CoA metabolic signaling pathway known to direct lipid partitioning toward oxidation and 2) increase the importing of fatty acids into the mitochondrion. These results suggest that T(2) stimulates mitochondrial fatty acid oxidation by activating several metabolic pathways, such as the fatty acid import/beta-oxidation cycle/FADH(2)-linked respiratory pathways, where fatty acids are imported. T(2) also enhanced skeletal muscle mitochondrial thermogenesis by activating pathways involved in the dissipation of the proton-motive force not associated with ATP synthesis (proton leak), the effect being dependent on the presence of free fatty acids inside mitochondria. We conclude that skeletal muscle is a target for T(2), and we propose that, by activating processes able to enhance mitochondrial fatty acid oxidation and thermogenesis, T(2) could play a role in protecting skeletal muscle against excessive intramyocellular lipid storage, possibly allowing it to avoid functional disorders.

Activation and inactivation of thyroid hormone by type I iodothyronine deiodinase

The prohormone thyroxine (T4) is activated by outer ring deiodination (ORD) to 3,3′,5‐triiodothyronine (T3) and both hormones are degraded by inner ring deiodination (IRD) to 3,3′,5′‐triiodothyronine (rT3) and 3,3′‐diiodothyronine, respectively. Indirect evidence suggests that the type I iodothyronine deiodinase (ID‐I) in liver has both ORD and IRD activities, with preference for rT3 and sulfated iodothyronines as substrates. To establish this, we have compared the ORD of rT3 and IRD of T3 and T3 sulfate by homogenates of cells transfected with rat ID‐I cDNA and by rat liver microsomes. In both preparations rT3 is the preferred substrate, while deiodination of T3 is markedly accelerated by its sulfation. Kinetic analysis provided similar K m and V max values in cell homogenates and liver microsomes. These data demonstrate unequivocally that ID‐I is capable of both activating and inactivating thyroid hormone by ORD and IRD, respectively.

Rapid stimulation in vitro of rat liver cytochrome oxidase activity by 3,5-diiodo-l-thyronine and by 3,3′-diiodo-l-thyronine

The effect of the iodothyronines (thyroxine (T4), 3,5,3-triiodo-L-thyronine (L-T3), 3,5-diiodo-L-thyronine (3,5-T2), 3,3-diiodo-L-thyronine (3,3-T2), 3,5-diiodo-L-thyronine (3,5-T2), 3-monoiodo-L-thyronine (3-T1), 3-monoiodo-L-thyronine (3-T1) and thyronine (T0)) on rat liver cytochrome oxidase (COX) activity after their addition to rat liver homogenate and isolated mitochondria from normal and hypothyroid rats has been investigated. The addition of 3,3-T2 and 3,5-T2 (T2s) to the liver homogenate from hypothyroid rats, but not from normal rats, significantly enhanced COX activity. The addition of T3 had a remarkably lower effect that was almost completely abolished when the propylthiouracil (PTU), an inhibitor of the type I deiodinase activity, was also added to the incubation mixture. After the addition of T2s the maximum effect was obtained at a concentration of about 10(-6) M for both 3,3-T2 and 3,5-T2, while a 50% increase was obtained at a concentration of about 10(-9) M in both cases. The effects of T2s were rapid and already evident after 5 min of incubation (+40-50%). The maximal effect was reached after only 30 min of incubation. The above effects were not observed after the addition of T2s to the isolated mitochondria. The results clearly demonstrate that both 3,3-T2 and 3,5-T2 directly stimulate mitochondrial COX activity which is possibly achieved through a cytoplasmic factor. The addition of the other iodothyronines (T4, 3,5-T2, 3-T1, 3-T1 and T0).

Effect of 3,3'-diiodothyronine and 3,5-diiodothyronine on rat liver oxidative capacity

We report that 3,5,3-triiodothyronine (T3) as well as two other iodothyronines (3,3-diiodothyronine and 3,5-diiodothyronine (T2s)) stimulate rat liver oxidative capacity (measured as cytochrome oxidase activity (COX)). In hypothyroid rats COX activity and mitochondrial protein content are significantly lower than in normal control animals. The administration of both T3 and T2s to hypothyroid rats significantly enhances hepatic COX activity with T3 having the greatest effect (+60%); moreover, T3 restores the mitochondrial protein content whereas the T2s are ineffective. Administration of T2s results in a faster stimulation (already significant 1 h after the injection) of hepatic COX activity than T3 injection. Our results suggest that T3 acts on the protein synthesis mechanism involved in the regulation of the mitochondrial mass while T2s would act directly at the mitochondrial level.

Segregation of the intra- and extrahypothalamic neuropeptide Y and catecholaminergic inputs on paraventricular neurons, including those producing thyrotropin-releasing hormone.

In fasting, declining circulating thyroid hormone levels coincide with suppressed thyrotropin-releasing hormone (TRH) mRNA and peptide levels and elevated NPY release and binding in the parvicellular paraventricular nucleus (PVN). It is suggested that NPY, in parallel with triggering feeding behavior, interrupts normal thyroid feedback in food deprivation. To gain further insights into the involvement of NPY in the regulation of TRH cells, this study sought to elucidate the source of the NPY innervation of TRH neurons. The median forebrain bundle (MFB) that carries the ascending NPY fibers from the brain stem catecholaminergic nuclei was unilaterally transected. Animals were sacrificed 2 and 5 days after surgery and double immunocytochemistry for NPY and TRH or tyrosine hydroxylase (TH) and TRH was performed on sections from the PVN. Two days after the surgery, light microscopic examination revealed no changes in the numbers of NPY boutons making putative contacts with TRH cell bodies and proximal dendrites. On the other hand, under the electron microscope, NPY- and TH-immunoreactive fibers containing autophagous cytolysosomes, an early sign of catecholaminergic fiber degeneration, were found to establish asymmetric synapses on distal dendrites and dendritic spines of TRH-immunoreactive cells. However, the same electron microscopic analysis did not reveal any degenerating NPY-immunolabeled fibers in synaptic contact with TRH cell bodies and proximal dendrites. Five days after the surgery, when NPY and TH immunoreactivities were no longer detected in the ipsilateral MFB, no decrease in the numbers of NPY and TH boutons on TRH cell bodies and proximal dendrites could be detected, when compared to the contralateral side. Electron microscopy revealed fibers with Wallerian degeneration establishing asymmetric synapses exclusively on the distal dendrites and spines of TRH neurons. In conclusion, this study demonstrated that the NPY and catecholaminergic input on PVN TRH cells are of mixed origin. The cell bodies and proximal dendrites of TRH neurons receive a robust, putative inhibitory NPY input from the hypothalamus. The distal dendrites and dendritic spines of the TRH cells also receive a putative stimulatory NPY input from the brain stem catecholaminergic neurons. It is suggested that because of its proximal location and abundance, NPY of hypothalamic origin exerts a tonic inhibition on PVN TRH cells that interrupts negative thyroid feedback during food deprivation. Furthermore, it is likely that a general inhibition and not stimulation of parvicellular PVN activity may underlie the triggering of feeding behavior by hypothalamic NPY.

De novo expression of uncoupling protein 3 is associated to enhanced mitochondrial thioesterase‐1 expression and fatty acid metabolism in liver of fenofibrate‐treated rats

Uncoupling protein 3 (UCP3) is a member of the mitochondrial carrier superfamily, preferentially expressed in skeletal muscle. Its function is not fully understood and it is debated whether it uncouples oxidative phosphorylation as does UCP1 in brown adipose tissue. Recent evidences suggest a role for UCP3 in the flux of fatty acids in and out mitochondria and their utilization in concert with mitochondrial thioesterase‐1 (MTE‐1). In fact, mice overexpressing muscle UCP3 also show high levels of MTE‐1. Fenofibrate is a hypolipidemic drug that prevents body weight gain in diet‐induced obese rats and enhances lipid metabolism by activating peroxisome proliferator‐activated receptors (PPARs). Because fatty acids and fenofibrate stimulate PPARs and in turn UCP3, we investigated whether UCP3 expression might be induced ‘de novo’ in situations of increased hepatic mitochondrial fatty acid utilization caused by a combined effect of a high‐fat diet and fenofibrate treatment. We also investigated whether Mte‐1 expression and β‐oxidation were affected. We show here that Ucp3 is induced in liver of fenofibrate‐treated rats at the mRNA and protein level. Expression was restricted to hepatocytes and was unevenly distributed in the liver. No increase in cell proliferation, inflammatory or fibrotic responses was found. Mte‐1 expression and mitochondrial β‐oxidation were upregulated. Thus, Ucp3 can be transactivated in tissues where it is normally silent and fenofibrate can attain this effect in liver. The data demonstrate that UCP3 is involved in fatty acid utilization and support the notion that UCP3 and MTE‐1 are linked within the same metabolic pathway.

Thyroid-hormone effects on putative biochemical pathways involved in UCP3 activation in rat skeletal muscle mitochondria.

In vitro, uncoupling protein 3 (UCP3)‐mediated uncoupling requires cofactors [e.g., superoxides, coenzyme Q (CoQ) and fatty acids (FA)] or their derivatives, but it is not yet clear whether or how such activators interact with each other under given physiological or pathophysiological conditions. Since triiodothyronine (T3) stimulates lipid metabolism, UCP3 expression and mitochondrial uncoupling, we examined its effects on some biochemical pathways that may underlie UCP3‐mediated uncoupling. T3‐treated rats (Hyper) showed increased mitochondrial lipid‐oxidation rates, increased expression and activity of enzymes involved in lipid handling and increased mitochondrial superoxide production and CoQ levels. Despite the higher mitochondrial superoxide production in Hyper, euthyroid and hyperthyroid mitochondria showed no differences in proton‐conductance when FA were chelated by bovine serum albumin. However, mitochondria from Hyper showed a palmitoyl–carnitine‐induced and GDP‐inhibited increased proton‐conductance in the presence of carboxyatractylate. We suggest that T3 stimulates the UCP3 activity in vivo by affecting the complex network of biochemical pathways underlying the UCP3 activation.