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Refeeding after caloric restriction induces weight regain and a disproportionate recovering of fat mass rather than lean mass (catch-up fat) that, in humans, associates with higher risks to develop chronic dysmetabolism. Studies in a well-established rat model of semistarvation-refeeding have reported that catch-up fat associates with hyperinsulinemia, glucose redistribution from skeletal muscle to white adipose tissue and suppressed adaptive thermogenesis sustaining a high efficiency for fat deposition. The skeletal muscle of catch-up fat animals exhibits reduced insulin-stimulated glucose utilization, mitochondrial dysfunction, delayed in vivo contraction-relaxation kinetics, increased proportion of slow fibers and altered local thyroid hormone metabolism, with suggestions of a role for iodothyronine deiodinases. To obtain novel insights into the skeletal muscle response during catch-up fat in this rat model, the functional proteomes of tibialis anterior and soleus muscles, harvested after 2 weeks of caloric restriction and 1 week of refeeding, were studied. Furthermore, to assess the implication of thyroid hormone metabolism in catch-up fat, circulatory thyroid hormones as well as liver type 1 (D1) and liver and skeletal muscle type 3 (D3) iodothyronine deiodinase activities were evaluated. The proteomic profiling of both skeletal muscles indicated catch-up fat-induced alterations, reflecting metabolic and contractile adjustments in soleus muscle and changes in glucose utilization and oxidative stress in tibialis anterior muscle. In response to caloric restriction, D3 activity increased in both liver and skeletal muscle, and persisted only in skeletal muscle upon refeeding. In parallel, liver D1 activity decreased during caloric restriction, and persisted during catch-up fat at a time-point when circulating levels of T4, T3 and rT3 were all restored to those of controls. Thus, during catch-up fat, a local hypothyroidism may occur in liver and skeletal muscle despite systemic euthyroidism. The resulting reduced tissue thyroid hormone bioavailability, likely D1- and D3-dependent in liver and skeletal muscle, respectively, may be part of the adaptive thermogenesis sustaining catch-up fat. These results open new perspectives in understanding the metabolic processes associated with the high efficiency of body fat recovery after caloric restriction, revealing new implications for iodothyronine deiodinases as putative biological brakes contributing in suppressed thermogenesis driving catch-up fat during weight regain. © Copyright © 2021 Di Munno, Busiello, Calonne, Salzano, Miles-Chan, Scaloni, Ceccarelli, de Lange, Lombardi, Senese, Cioffi, Visser, Peeters, Dulloo and Silvestri.

3,5-diiodo-thyronine (T2), an endogenous metabolite of thyroid hormones, exerts beneficial metabolic effects. When administered to overweight rats receiving a high fat diet (HFD), it significantly reduces body fat accumulation, which is a risk factor for the development of an inflammatory state and of related metabolic diseases. In the present study, we focused our attention on T2 actions aimed at improving the adverse effects of long-lasting HFD such as the adipocyte inflammatory response. For this purpose, three groups of rats were used throughout: i) receiving a standard diet for 14 weeks; ii) receiving a HFD for 14 weeks, and iii) receiving a HFD for 14 weeks with a simultaneous daily injection of T2 for the last 4 weeks. The results showed that T2 administration ameliorated the expression profiles of pro- and anti-inflammatory cytokines, reduced macrophage infiltration in white adipose tissue, influenced their polarization and reduced lymphocytes recruitment. Moreover, T2 improved the expression of hypoxia markers, all altered in HFD rats, and reduced angiogenesis by decreasing the pro-angiogenic miR126 expression. Additionally, T2 reduced the oxidative damage of DNA, known to be associated to the inflammatory status. This study demonstrates that T2 is able to counteract some adverse effects caused by a long-lasting HFD and to produce beneficial effects on inflammation. Irisin and SIRT1 pathway may represent a mechanism underlying the above described effects.

Fasting enhances the beneficial metabolic outcomes of exercise; however, it is unknown whether body composition is favorably modified on the short term. A baseline–follow-up study was carried out to assess the effect of an established protocol involving short-term combined exercise with fasting on body composition. One hundred seven recreationally exercising males underwent a 10-day intervention across 15 fitness centers in the Netherlands involving a 3-day gradual decrease of food intake, a 3-day period with extremely low caloric intake, and a gradual 4-day increase to initial caloric intake, with daily 30-min submaximal cycling. Using dual-energy X-ray absorptiometry analysis, all subjects substantially lost total body mass (−3.9 ± 1.9 kg; p p p p p p

Exercise under fasting conditions induces a switch to lipid metabolism, eliciting beneficial metabolic effects. Knowledge of signaling responses underlying metabolic adjustments in such conditions may help to identify therapeutic strategies. Therefore, we studied the effect of mild exercise on rats submitted to food withdrawal at thermoneutrality (28°C) for 3 days. Animals were housed at thermoneutrality rather than the standard housing temperature (22°C) to avoid beta‐adrenergic signaling responses that themselves affect metabolism and well‐being. Quantitative analysis of multi‐organ mRNA levels, myofibers, and serum metabolites shows that this protocol (a) boosts fat oxidation in muscle and liver, (b) reduces lipogenesis and increases gluconeogenesis in liver, (c) increases serum acylcarnitines (especially C4OH) and ketone bodies and the use of the latter as fuel in muscle, (d) increases Type I myofibers, and (e) is associated with an increased thyroid hormone uptake and metabolism in muscle. In addition, stool microbiome DNA analysis revealed that food withdrawal dramatically alters the presence of bacterial genera associated with ketone metabolism. Taken together, this protocol induces a drastic switch toward increased lipid and ketone metabolism compared to exercise or food withdrawal alone, which may prove beneficial and may involve local thyroid hormones, which may be regarded as exercise mimetics., Environmental temperature influences metabolic responses. The results of the present study show that the relatively mild metabolic effects of exercise at thermoneutrality are boosted when combined with food withdrawal. To our knowledge, this is the first study to reveal this, studying the whole animal's response ranging from microbiota to muscle, liver, and serum, suggesting a contributing role for thyroid hormone.

The present investigation was undertaken to increase our insight into the molecular basis of the physiological changes in rat testis induced by food withdrawal, and to clarify whether reduced testicular function can be ameliorated by mild exercise. Male rats were selected for four separate experiments. The first of each group was chow-fed, the second was chow-fed and submitted to exercise (5 bouts in total for 30 min at 15 m/min, and 0° inclination), the third was submitted to food withdrawal (66 h) and the fourth was submitted to food withdrawal and to exercise. At the end of experiments, we investigated (i) serum and testicular sex hormone levels; (ii) protein levels of StAR, 3β-Hydroxysteroid dehydrogenase (3β-HSD) and P450 aromatase, which play a key role in steroid hormone biosynthesis; and (iii) protein levels of mitotic and meiotic markers of spermatogenesis in rats, in relation to testis morphology and morphometry. We found that mild exercise or food withdrawal alone induced a significant increase or decrease in both serum and testis testosterone levels, respectively. Interestingly, we found that these levels were brought back to basal levels when food withdrawal was combined with mild exercise. The changes in testosterone levels observed in our experimental groups correlated well with the expression of steroidogenic enzymes as well as with spermatogenic activity. With mild exercise the increased testosterone/17β-estradiol (T/E2) ratio in the testis correlated with an increased spermatogenic activity. The T/E2 ratio dropped in fasted rats and was significantly reversed when food withdrawal was combined with exercise. Histological and morphometric analyses confirmed that spermatogenic activity varied in concomitance with each experimental condition. Importantly, the testis and serum T/E2 ratios correlated, confirming that exercise rescues the decline in food withdrawal-induced spermatogenesis. In conclusion, this study highlights that mild exercise normalizes the reduced spermatogenic activity caused by food withdrawal through the modulation of the steroidogenic pathway and restoring the T/E2 ratio, underlining the beneficial effects of mild exercise on the prevention and/or amelioration of reduced testis function caused by restricted caloric intake.

Both 3,5-diiodo-L-thyronine (3,5-T2) and 3,5,3'-triiodo-L-tyronine (T3) affect energy metabolism having mitochondria as a major target. However, the underlying mechanisms are poorly understood. Here, using a model of chemically induced hypothyroidism in male Wistar rats, we investigated the effect of administration of either 3,5-T2 or T3 on liver oxidative capacity through their influence on mitochondrial processes including: proton-leak across the mitochondrial inner membrane; complex I-, complex II- and glycerol-3-phosphate-linked respiratory pathways; respiratory complex abundance and activities as well as individual complex aggregation into supercomplexes. Background/Aims: Both 3,5-diiodo-L-thyronine (3,5-T2) and 3,5,3'-triiodo-L-tyronine (T3) affect energy metabolism having mitochondria as a major target. However, the underlying mechanisms are poorly understood. Here, using a model of chemically induced hypothyroidism in male Wistar rats, we investigated the effect of administration of either 3,5-T2 or T3 on liver oxidative capacity through their influence on mitochondrial processes including: proton-leak across the mitochondrial inner membrane; complex I-, complex II- and glycerol-3-phosphate-linked respiratory pathways; respiratory complex abundance and activities as well as individual complex aggregation into supercomplexes. Methods: Hypothyroidism was induced by propylthiouracil and iopanoic acid; 3,5-T2 and T3 were intraperitoneally administered at 25 and 15 μg/100 g BW for 1 week, respectively. Resulting alterations in mitochondrial function were studied by combining respirometry, Blue Native-PAGE followed by in-gel activity, and Western blot analyses. Results: Administration of 3,5-T2 and T3 to hypothyroid (hypo) rats enhanced mitochondrial respiration rate with only T3 effectively stimulating proton-leak (450% vs. Hypo). T3 significantly enhanced complex I (+145% vs. Hypo), complex II (+66% vs. Hypo), and glycerol-3 phosphate dehydrogenase (G3PDH)-linked oxygen consumptions (about 6- fold those obtained in Hypo), while 3,5-T2 administration selectively restored Euthyroid values of complex II- and increased G3PDH- linked respiratory pathways (+165% vs. Hypo). The mitochondrial abundance of all respiratory complexes and of G3PDH was increased by T3 administration whereas 3,5-T2 only increased complex V and G3PDH abundance. 3,5-T2 enhanced complex I and complex II in gel activities with less intensity than did T3, and T3 also enhanced the activity of all other respiratory complexes tested. In addition, only T3 enhanced individual respiratory component complex assembly into supercomplexes. Conclusions: The reported data highlight novel molecular mechanisms underlying the effect elicited by iodothyronine administration to hypothyroid rats on mitochondrial processes related to alteration in oxidative capacity in the liver. The differential effects elicited by the two iodothyronines indicate that 3,5-T2, by influencing the kinetic properties of specific mitochondrial respiratory pathways, would promote a rapid response of the organelle, while T3, by enhancing the abundance of respiratory chain component and favoring the organization of respiratory chain complex in supercomplexes, would induce a slower and prolonged response of the organelle.

Hyperlipidemic state-associated perturbations in the network of factors controlling mitochondrial functions, i.e., morphogenesis machinery and metabolic sensor proteins, produce metabolic inflexibility, insulin resistance and reduced oxidative capacity in skeletal muscle. Moreover, intramyocellular lipid (IMCL) accumulation leads to tissue damage and inflammation. The administration of the naturally occurring metabolite 3,5-diiodo-L-thyronine (T2) with thyromimetic actions to high fat diet (HFD)-fed rats exerts a systemic hypolipidemic effect, which produces a lack of IMCL accumulation, a shift toward glycolytic fibers and amelioration of insulin sensitivity in gastrocnemius muscle. In this study, an integrated approach combining large-scale expression profile and functional analyses was used to characterize the response of skeletal muscle mitochondria to 12 during a HFD regimen. Long-term 12 administration to HDF rats induced a glycolytic phenotype of gastrocnemius muscle as well as an adaptation of mitochondria to the fiber type, with a decreased representation of enzymes involved in mitochondrial oxidative metabolism. At the same time, T2 stimulated the activity of individual respiratory complex I, IV and V. Moreover, 12 prevented the HFD-associated increase in the expression of peroxisome proliferative activated receptor gamma coactivator-1 alpha and dynamin-1-like protein as well as mitochondrial morphological aberrations, favoring the appearance of tubular and tethered organelles in the intermyofibrillar regions. Remarkably, 12 reverted the HDF-associated expression pattern of proinflammatory factors, such as p65 subunit of NF-kB, and increased the fiber-specific immunoreactivity of adipose differentiation related protein in lipid droplets. All together, these results further support a role of T2 in counteracting in vivo some of the HFD-induced impairment in structural/metabolic features of skeletal muscle by impacting the mitochondrial phenotype.

The role of uncoupling protein-3 (UCP3) in energy and lipid metabolism was investigated. Male wild-type (WT) and UCP3-null (KO) mice that were housed at thermoneutrality (30 &deg, C) were used as the animal model. In KO mice, the ability of skeletal muscle mitochondria to oxidize fatty acids (but not pyruvate or succinate) was reduced. At whole animal level, adult KO mice presented blunted resting metabolic rates, energy expenditure, food intake, and the use of lipids as metabolic substrates. When WT and KO mice were fed with a standard/low-fat diet for 80 days, since weaning, they showed similar weight gain and body composition. Interestingly, KO mice showed lower fat accumulation in visceral adipose tissue and higher ectopic fat accumulation in liver and skeletal muscle. When fed with a high-fat diet for 80 days, since weaning, KO mice showed enhanced energy efficiency and an increased lipid gain (thus leading to a change in body composition between the two genotypes). We conclude that UCP3 plays a role in energy and lipid homeostasis and in preserving lean tissues by lipotoxicity, in mice that were housed at thermoneutrality.

This study evaluated the effect of 3,5-diiodo-L-thyronine (T2) and 3,5,3′-triiodo-L-thyronine (T3) on rat liver mitochondrial DNA (mtDNA) oxidative damage and repair and to investigate their ability to induce protective effects against oxidative stress. Control rats, rats receiving a daily injection of T2 (N+T2) for 1 week and rats receiving a daily injection of T3 (N+T3) for 1 week, were used throughout the study. In the liver, mtDNA oxidative damage [by measuring mtDNA lesion frequency and expression of DNA polymerase γ (POLG)], mtDNA copy number, mitochondrial biogenesis [by measuring amplification of mtDNA/nDNA and expression of peroxisome proliferator-activated receptor gamma co-activator 1-alpha (PGC-1α)], and oxidative stress [by measuring serum levels of 8-hydroxy-2′-deoxyguanosine (8-OHdG)] were detected. T2 reduces mtDNA lesion frequency and increases the expression of POLG, and it does not change the mtDNA copy number, the expression of PGC-1α, or the serum levels of 8-OHdG. Therefore, T2, by stimulating the major mtDNA repair enzyme, maintains genomic integrity. Similar to T2, T3 decreases mtDNA lesion frequency but increases the serum levels of 8-OHdG, and it decreases the expression of POLG. Moreover, as expected, T3 increases the mtDNA copy number and the expression of PGC-1α. Thus, in T3-treated rats, the increase of 8-OHdG and the decrease of POLG indicate that there is increased oxidative damage and that the decreased mtDNA lesion frequency might be a consequence of increased mitochondrial biogenesis. These data demonstrate that both T2 and T3 are able to decrease in the liver mtDNA oxidative damage, but they act via different mechanisms.

The conversion of white adipose cells into beige adipose cells is known as browning, a process affecting energy metabolism. It has been shown that 3,5 diiodo-l-thyronine (T2), an endogenous metabolite of thyroid hormones, stimulates energy expenditure and a reduction in fat mass. In light of the above, the purpose of this study was to test whether in an animal model of fat accumulation, T2 has the potential to activate a browning process and to explore the underlying mechanism. Three groups of rats were used: (i) receiving a standard diet for 14 weeks, (ii) receiving a high-fat diet (HFD) for 14 weeks, and (iii) receiving a high fat diet for 10 weeks and being subsequently treated for four weeks with an HFD together with the administration of T2. We showed that T2 was able to induce a browning in the white adipose tissue of T2-treated rats. We also showed that some miRNA (miR133a and miR196a) and MAP kinase 6 were involved in this process. These results indicate that, among others, the browning may be another cellular/molecular mechanism by which T2 exerts its beneficial effects of contrast to overweight and of reduction of fat mass in rats subjected to HFD.

When administered to rats receiving a high-fat diet (HFD), 3,5-diiodo-L-thyronine (3,5-T2) [at a dose of 25 &mu, g/100 g body weight (BW)] is known to increase energy expenditure and to prevent HFD-induced adiposity. Here, we investigated which cellular and molecular processes in visceral white adipose tissue (VAT) contributed to the beneficial effect of 3,5-T2 over time (between 1 day and 4 weeks following administration). 3,5-T2 programmed the adipocyte for lipolysis by rapidly inducing hormone sensitive lipase (HSL) phosphorylation at the protein kinase A-responsive site Ser563, accompanied with glycerol release at the 1-week time-point, contributing to the partial normalization of adipocyte volume with respect to control (N) animals. After two weeks, when the adipocyte volumes of HFD-3,5-T2 rats were completely normalized to those of the controls (N), 3,5-T2 consistently induced HSL phosphorylation at Ser563, indicative of a combined effect of 3,5-T2-induced adipose lipolysis and increasing non-adipose oxidative metabolism. VAT proteome analysis after 4 weeks of treatment revealed that 3,5-T2 significantly altered the proteomic profile of HFD rats and produced a marked pro-angiogenic action. This was associated with a reduced representation of proteins involved in lipid storage or related to response to oxidative stress, and a normalization of the levels of those involved in lipogenesis-associated mitochondrial function. In conclusion, the prevention of VAT mass-gain by 3,5-T2 occurred through different molecular pathways that, together with the previously reported stimulation of resting metabolism and liver fatty acid oxidation, are associated with an anti adipogenic/lipogenic potential and positively impact on tissue health.

Over 30 years of research has demonstrated that 3,5-diiodo-L-thyronine (3,5-T2), an endogenous metabolite of thyroid hormones, exhibits interesting metabolic activities. In rodent models, exogenously administered 3,5-T2 rapidly increases resting metabolic rate and elicits short-term beneficial hypolipidemic effects; however, very few studies have evaluated the effects of endogenous and exogenous T2 in humans. Further analyses on larger cohorts are needed to determine whether 3,5-T2 is a potent additional modulator of energy metabolism. In addition, while several lines of evidence suggest that 3,5-T2 mainly acts through Thyroid hormone receptors (THRs)- independent ways, with mitochondria as a likely cellular target, THRs-mediated actions have also been described. The detailed cellular and molecular mechanisms through which 3,5-T2 elicits a multiplicity of actions remains unknown. Here, we provide an overview of the most recent literature on 3,5-T2 bioactivity with a particular focus on short-term and long-term effects, describing data obtained through in vivo and in vitro approaches in both mammalian and non-mammalian species. Over 30 years of research has demonstrated that 3,5-diiodo-L-thyronine (3,5-T2), an endogenous metabolite of thyroid hormones, exhibits interesting metabolic activities. In rodent models, exogenously administered 3,5-T2 rapidly increases resting metabolic rate and elicits short-term beneficial hypolipidemic effects; however, very few studies have evaluated the effects of endogenous and exogenous T2 in humans. Further analyses on larger cohorts are needed to determine whether 3,5-T2 is a potent additional modulator of energy metabolism. In addition, while several lines of evidence suggest that 3,5-T2 mainly acts through Thyroid hormone receptors (THRs)-independent ways, with mitochondria as a likely cellular target, THRs-mediated actions have also been described. The detailed cellular and molecular mechanisms through which 3,5-T2 elicits a multiplicity of actions remains unknown. Here, we provide an overview of the most recent literature on 3,5-T2 bioactivity with a particular focus on short-term and long-term effects, describing data obtained through in vivo and in vitro approaches in both mammalian and non-mammalian species.

3,3′,5-triiodo-L-thyronine (T3) improves hepatic lipid accumulation by increasing lipid catabolism but it also increases lipogenesis, which at first glance appears contradictory. Recent studies have shown that 3,5-diiodothyronine (T2), a natural thyroid hormone derivative, also has the capacity to stimulate hepatic lipid catabolism, however, little is known about its possible effects on lipogenic gene expression. Because genes classically involved in hepatic lipogenesis such as SPOT14, acetyl-CoA-carboxylase (ACC), and fatty acid synthase (FAS) contain thyroid hormone response elements (TREs), we studied their transcriptional regulation, focusing on TRE-mediated effects of T3 compared to T2 in rats receiving high-fat diet (HFD) for 1 week. HFD rats showed a marked lipid accumulation in the liver, which was significantly reduced upon simultaneous administration of either T3 or T2 with the diet. When administered to HFD rats, T2, in contrast with T3, markedly downregulated the expression of the above-mentioned genes. T2 downregulated expression of the transcription factors carbohydrate-response element-binding protein (ChREBP) and sterol regulatory element binding protein-1c (SREBP-1c) involved in activation of transcription of these genes, which explains the suppressed expression of their target genes involved in lipogenesis. T3, however, did not repress expression of the TRE-containing ChREBP gene but repressed SREBP-1c expression. Despite suppression of SREBP-1c expression by T3 (which can be explained by the presence of nTRE in its promoter), the target genes were not suppressed, but normalized to HFD reference levels or even upregulated (ACC), partly due to the presence of TREs on the promoters of these genes and partly to the lack of suppression of ChREBP. Thus, T2 and T3 probably act by different molecular mechanisms to achieve inhibition of hepatic lipid accumulation.

Thyroid hormones (THs) are produced by the thyroid gland and converted in peripheral organs by deiodinases. THs regulate cell functions through two distinct mechanisms: genomic (nuclear) and nongenomic (non-nuclear). Many TH effects are mediated by the genomic pathway – a mechanism that requires TH activation of nuclear thyroid hormone receptors. The overall nongenomic processes, emerging as important accessory mechanisms in TH actions, have been observed at the plasma membrane, in the cytoplasm and cytoskeleton, and in organelles. Some products of peripheral TH metabolism (besides triiodo-l-thyronine), now termed ‘nonclassical THs’, were previously considered as inactive breakdown products. However, several reports have recently shown that they may have relevant biological effects. The recent accumulation of knowledge on how classical and nonclassical THs modulate the activity of membrane receptors, components of the mitochondrial respiratory chain, kinases and deacetylases, opened the door to the discovery of new pathways through which they act. We reviewed the current state-of-the-art on the actions of the nonclassical THs, discussing the role that these endogenous TH metabolites may have in the modulation of thyroid-related effects in organisms with differing complexity, ranging from nonmammals to humans.

The activity of the thyroid gland diminishes during ageing, but a certain tissue reserve of T3 and its metabolites is maintained. This reserve is thought to play a regulatory role in energy homeostasis during ageing. This review critically assesses this notion. T3 was thought to act predominantly through pathways that require transcriptional regulation by thyroid hormone receptors (TRs). However, in recent years, it has emerged that T3 and its metabolites can also act through non-genomic mechanisms, including cytosolic signaling. Interestingly, differences may exist in the non-genomic pathways utilized by thyroid hormone metabolites and T3. For instance, one particular thyroid hormone metabolite, namely 3,5-diiodo-L-thyronine (T2), increases the activity of the redox-sensitive protein deacetylase SIRT1, which has been associated with improvements in healthy ageing, whereas evidence exists that T3 may have the opposite effect. Findings suggesting that T3, T2, and their signaling pathways, such as those involving SIRT1 and AMP-activated protein kinase (AMPK), are associated with improvements in diet-induced obesity and insulin resistance emphasize the potential importance of the thyroid during ageing and in ageing-associated metabolic diseases. © 2013 by the authors; licensee MDPI, Basel, Switzerland.

Obesity and type 2 diabetes are associated disorders that involve a multiplicity of tissues. Both fasting and physical exercise are known to counteract dyslipidemia/hyperglycemia. Skeletal muscle plays a key role in the control of blood glucose levels, and the metabolic changes and related signaling pathways in skeletal muscle induced by fasting overlap with those induced by exercise. The reduction of fat disposal has been shown to extend to the liver and to white and brown adipose tissue and to involve an increase in their metabolic activities. In recent years signal transduction pathways related to exercise and fasting/food withdrawal in muscle have been intensively studied, both in animals and in humans. Combining fasting/food withdrawal with exercise in animals as well as in humans causes changes unlike those seen during fasting/food withdrawal or exercise alone, which favor repair of muscle over autophagy. In addition, compounds that mimic exercise have been studied in combination with exercise or fasting/food withdrawal. This review addresses our current knowledge of the mechanisms that underlie the individual and combined effects of fasting/food withdrawal, endurance or resistance exercise, and their mimetics, in muscle vs other organs in rodents and humans, and highlights which combinations may improve metabolic disorders.-Jaspers, R. T., Zillikens, M. C., Friesema, E. C. H., delli Paoli, G., Bloch, W., Uitterlinden, A. G., Goglia, F., Lanni, A., de Lange, P. Exercise, fasting, and mimetics: toward beneficial combinations. Obesity and type 2 diabetes are associated disorders that involve a multiplicity of tissues. Both fasting and physical exercise are known to counteract dyslipidemia/hyperglycemia. Skeletal muscle plays a key role in the control of blood glucose levels, and the metabolic changes and related signaling pathways in skeletal muscle induced by fasting overlap with those induced by exercise. The reduction of fat disposal has been shown to extend to the liver and to white and brown adipose tissue and to involve an increase in their metabolic activities. In recent years signal transduction pathways related to exercise and fasting/food withdrawal in muscle have been intensively studied, both in animals and in humans. Combining fasting/food withdrawal with exercise in animals aswell as in humans causes changes unlike those seen during fasting/food withdrawal or exercise alone, which favor repair of muscle over autophagy. In addition, compounds that mimic exercise have been studied in combination with exercise or fasting/food with drawal. This review addresses our current knowledge of the mechanisms that underlie the individual and combined effects of fasting/food withdrawal, endurance or resistance exercise, and their mimetics, in muscle vs. other organs in rodents and humans, and highlights which combinations may improve metabolic disorders. -Jaspers, R. T., Zillikens, M. C., Friesema, E. C. H., delli Paoli, G., Bloch, W., Uitterlinden, A. G., Goglia, F., Lanni, A., de Lange, P. Exercise, fasting, and mimetics: toward beneficial combinations.

Chronic overnutrition and modern life-styles are causing a worldwide epidemic of obesity and associated comorbidities, which is creating a demand to identify underlying biological mechanisms and to devise effective treatments. In rats receiving a high-fat diet (HFD), we analyzed the effects of a 4-wk administration of a novel functional analog of iodothyronines, TRC150094 (TRC). HFD-TRC rats exhibited increased energy expenditure (+24% vs. HFD rats; P

T3 stimulates metabolic rate in many tissues and induces changes in fuel use. The pathways by which T3 induces metabolic/structural changes related to altered fuel use in skeletal muscle have not been fully clarified. Gastrocnemius muscle (isolated at different time points after a single injection of T3 into hypothyroid rats), displayed rapid inductions of AMP-activated protein kinase (AMPK) phosphorylation (threonine 172; within 6 h) and acetyl-coenzyme A carboxylase phosphorylation (serine 79; within 12 h). As a consequence, increases occurred in mitochondrial fatty acid oxidation and carnitine palmitoyl transferase activity. Concomitantly, T 3 stimulated signaling toward increased glycolysis through a rapid increase in Akt/protein kinase B (serine 473) phosphorylation (within 6 h) and a directly related increase in the activity of phosphofructokinase. The kinase specificity of the above effects was verified by treatment with inhibitors of AMPK and Akt activity (compound C and wortmannin, respectively). In contrast, glucose transporter 4 translocation to the membrane (activated by T3 within 6 h) was maintained when either AMPK or Akt activity was inhibited. The metabolic changes were accompanied by a decline in myosin heavy-chain Ib protein [causing a shift toward the fast-twitch (glycolytic) phenotype]. The increases in AMPK and acetyl-coenzyme A carboxylase phosphorylation were transient events, both levels declining from 12 h after the T3 injection, but Akt phosphorylation remained elevated until at least 48h after the injection. These data show that in skeletal muscle, T3 stimulates both fatty acid and glucose metabolism through rapid activations of the associated signaling pathways involving AMPK and Akt/protein kinase B. Copyright © 2008 by The Endocrine Society.

Aging is associated with changes in thyroid gland physiology. Age-related changes in the contribution of peripheral tissues to thyroid hormone serum levels have yet to be systematically assessed. Here, we investigated age-related alterations in the contributions of the liver and kidney to thyroid hormone homeostasis using 6-, 12-, and 24-mo-old male Wistar rats. A significant and progressive decline in plasma thyroxine occurred with age, but triiodothyronine (T3) was decreased only at 24 mo. This was associated with an unchanged protein level of the thyroid hormone transporter monocarboxylate transporter 8 (MCT8) in the kidney and with a decreased MCT8 level in the liver at 24 mo. Hepatic type I deiodinase (D1) protein level and activity declined progressively with age. Renal D1 levels were decreased at both 12 and 24 mo but D1 activity was decreased only at 24 mo. In the liver, no changes occurred in thyroid hormone receptor (TR) TRα1, whereas a progressive increase in TRβ1 occurred at both mRNA and total protein levels. In the kidney, both TRα1 and TRβ1 mRNA and total protein levels were unchanged between 6 and 12 mo but increased at 24 mo. Interestingly, nuclear TRβ1 levels were decreased in both liver and kidney at 12 and 24 mo, whereas nuclear TRα1 levels were unchanged. Collectively, our data show differential age-related changes among hepatic and renal MCT8 and D1 and TR expressions, and they suggest that renal D1 activity is maintained with age to compensate for the decrease in hepatic T3 production.

We investigated the mechanism by which 3,5-diiodo-l-thyronine (T2) affects skeletal muscle mitochondrial bioenergetic parameters following its acute administration to hypothyroid rats. One hour after injection, T2 increased both coupled and uncoupled respiration rates by +27% and +42%, respectively. Top-down elasticity analysis revealed that these effects were the result of increases in the substrate oxidation and mitochondrial uncoupling. Discriminating between proton-leak and redox-slip processes, we identified an increased mitochondrial proton conductance as the "pathway" underlying the effect of T2 on mitochondrial uncoupling. As a whole, these results may provide a mechanism by which T2 rapidly affects energy metabolism in hypothyroid rats. © 2007 Federation of European Biochemical Societies.

3,5,3′-Triiodo-L-thyronine (T3) plays a crucial role in regulating metabolic rate and fuel oxidation; however, the mechanisms by which it affects whole-body energy metabolism are still not completely understood. Skeletal muscle (SKM) plays a relevant role in energy metabolism and responds to thyroid state by remodeling the metabolic characteristics and cytoarchitecture of myocytes. These processes are coordinated with changes in mitochondrial content, bioenergetics, substrate oxidation rate, and oxidative phosphorylation efficiency. Recent data indicate that “emerging” iodothyronines have biological activity. Among these, 3,5-diiodo-L-thyronine (T2) affects energy metabolism, SKM substrate utilization, and mitochondrial functionality. The effects it exerts on SKM mitochondria involve more aspects of mitochondrial bioenergetics; among these, respiratory chain activity, mitochondrial thermogenesis, and lipid-handling are stimulated rapidly. This mini review focuses on signaling and biochemical pathways activated by T3 and T2 in SKM that influence the above processes. These novel aspects of thyroid physiology could reveal new perspectives for understanding the involvement of SKM mitochondria in hypo- and hyper-thyroidism.

Weight loss due to negative energy balance is a goal in counteracting obesity and type 2 diabetes mellitus. The thyroid is known to be an important regulator of energy metabolism through the action of thyroid hormones (THs). The classic, active TH, 3,5,3’-triiodo-L-thyronine (T3) acts predominantly by binding to nuclear receptors termed TH receptors (TRs), that recognize TH response elements (TREs) on the DNA, and so regulate transcription. T3 also acts through non-genomic pathways that do not necessarily involve TRs. Lipid-lowering therapies have been suggested to have potential benefits, however, the establishment of comprehensive therapeutic strategies is still awaited. One drawback of using T3 in counteracting obesity has been the occurrence of heart rhythm disturbances. These are mediated through one TR, termed TR alpha. The end of the previous century saw the exploration of TH mimetics that specifically bind to TR beta in order to prevent cardiac disturbances, and TH derivatives such as 3,5-diiodo-L-thyronine (T2), that possess interesting biological activities. Several TH derivatives and functional analogs have low affinity for the TRs, and are suggested to act predominantly through non-genomic pathways. All this has opened new perspectives in thyroid physiology and TH derivative usage as anti-obesity therapies. This review addresses the pros and cons of these compounds, in light of their effects on energy balance regulation and on lipid/cholesterol metabolism.

The effects of T3 administration on the rat Harderian gland were examined at morphological, biochemical, and molecular levels. T3 induced hypertrophy of the two cell types (A and B) present in the glandular epithelium. In type A cells, the hypertrophy was mainly due to an increase in the size of the lipid compartment. The acinar lumina were filled with lipoproteic substances, and the cells often showed an olocrine secretory pattern. In type B cells, the hypertrophy largely consisted of a marked proliferation of mitochondria endowed with tightly packed cristae, the mitochondrial number being nearly doubled (from 62 to 101/100 μm2). Although the average area of individual mitochondria decreased by about 50%, the total area of the mitochondrial compartment increased by about 80% (from 11 to 19/100 μm2). This could be ascribed to T3-induced mitochondrial proliferation. The morphological and morphometric data correlated well with our biochemical results, which indicated that mitochondrial respiratory activity is increased in hyperthyroid rats. T3, by influencing the metabolic function of the mitochondrial compartment, induces lipogenesis and the release of secretory product by type A cells. Mitochondrial uncoupling proteins 2 and 3 were expressed at both mRNA and protein levels in the euthyroid rat Harderian gland. T3 treatment increased the mRNA levels of both uncoupling protein 2 (UCP2) and UCP3, but the protein level only of UCP3. A possible role for these proteins in the Harderian gland is discussed.

Corticotropinomas are characterized by a relative resistance to the negative feedback action of cortisol on ACTH secretion. In this respect there is a similarity with the clinical syndrome of cortisol resistance. As cortisol resistance can be caused by genetic abnormalities in the glucocorticoid receptor (GR) gene, we investigated whether the insensitivity of corticotropinomas to cortisol is also caused by de novo mutations in the GR gene. We screened for the GR gene in leukocyte and tumor DNA from 22 patients with Cushing’s disease for mutations using PCR/single strand conformation polymorphism analysis. In a previous study, we identified 5 polymorphisms in the GR gene in a normal population. These polymorphisms were used as markers for the possible occurrence of loss of heterozygosity (LOH) at the GR gene locus. Except for 1 silent point mutation, we did not identify novel mutations in the GR gene in leukocytes or corticotropinomas from these patients. Of the 22 patients, 18 were heterozygous for at least 1 of the polymorphisms. In 6 of these patients, LOH had occurred in the tumor DNA. Of 21 patients examined for LOH on chromosome 11q13, only 1, with a corticotroph carcinoma, showed allelic deletion. As controls we studied 28 pituitary tumors of other subtypes (11 clinically nonfunctioning, 8 prolactinomas, and 9 GH-producing adenomas) and found evidence for LOH in only 1 prolactinoma. In six patients LOH was found at the GR gene locus (chromosome 5) in DNA derived from adenoma cells. Our observations indicate for the first time that LOH at the GR gene locus is a relatively frequent phenomenon in pituitary adenomas of patients with Cushing’s disease. This might explain the relative resistance of the adenoma cells to the inhibitory feedback action of cortisol on ACTH secretion. The specificity of the GR LOH to corticotropinomas supports this concept. Somatic mutations of the GR are not a frequent cause of relative cortisol resistance in these cells.

The molecular mechanisms underlying primary glucocorticoid resistance or hypersensitivity are not well understood. Using transfected COS-1 cells as a model system, we studied gene regulation by naturally occurring mutants of the glucocorticoid receptor (GR) with single-point mutations in the regions encoding the ligand-binding domain or the N-terminal domain reflecting different phenotypic expression. We analyzed the capacity of these GR variants to regulate transcription from different promoters, either by binding directly to positive or negative glucocorticoid-response elements on the DNA or by interfering with protein-protein interactions. Decreased dexamethasone (DEX) binding to GR variants carrying mutations in the ligand-binding domain correlated well with decreased capacity to activate transcription from the mouse mammary tumor virus (MMTV) promoter. One variant, D641V, which suboptimally activated MMTV promoter-mediated transcription, repressed a PRL promoter element containing a negative glucocorticoid-response element with wild type activity. DEX-induced repression of transcription from elements of the intercellular adhesion molecule-1 promoter via nuclear factor-kappaB by the D641V variant was even more efficient compared with the wild type GR. We observed a general DEX-responsive AP-1-mediated transcriptional repression of the collagenase-1 promoter, even when receptor variants did not activate transcription from the MMTV promoter. Our findings indicate that different point mutations in the GR can affect separate pathways of gene regulation in a differential fashion, which can explain the various phenotypes observed.

A novel functional iodothyronine analogue, TRC150094, which has a much lower potency toward thyroid hormone receptor (α1/β1) activation than triiodothyronine, has been shown to be effective at reducing adiposity in rats simultaneously receiving a high-fat diet (HFD). Here, by combining metabolic, functional and proteomic analysis, we studied how the hepatic and skeletal muscle phenotypes might respond to TRC150094 treatment in HFD-fed overweight rats. Drug treatment increased both the liver and skeletal muscle mitochondrial oxidative capacities without altering mitochondrial efficiency. Coherently, in terms of individual respiratory in-gel activity, blue-native analysis revealed an increased activity of complex V in the liver and of complexes II and V in tibialis muscle in TCR150094-treated animals. Subsequently, the identification of differentially expressed proteins and the analysis of their interrelations gave an integrated view of the phenotypic/metabolic adaptations occurring in the liver and muscle proteomes during drug treatment. TRC150094 significantly altered the expression of several proteins involved in key liver metabolic pathways, including amino acid and nitrogen metabolism, and fructose and mannose metabolism. The canonical pathways most strongly influenced by TRC150094 in tibialis muscle included glycolysis and gluconeogenesis, amino acid, fructose and mannose metabolism, and cell signaling. The phenotypic/metabolic influence of TRC150094 on the liver and skeletal muscle of HFD-fed overweight rats suggests the potential clinical application of this iodothyronine analogue in ameliorating metabolic risk parameters altered by diet regimens. © 2012 The Royal Society of Chemistry.

Iodothyronines such as triiodothyronine (T3) and 3,5-diiodothyronine (T2) influence energy expenditure and lipid metabolism. Skeletal muscle contributes significantly to energy homeostasis, and the above iodothyronines are known to act on this tissue. However, little is known about the cellular/molecular events underlying the effects of T3and T2on skeletal muscle lipid handling. Since FAT/CD36 is involved in the utilization of free fatty acids by skeletal muscle, specifically in their import into that tissue and presumably their oxidation at the mitochondrial level, we hypothesized that related changes in lipid handling and in FAT/CD36 expression and subcellular redistribution would occur due to hypothyroidism and to T3or T2administration to hypothyroid rats. In gastrocnemius muscles isolated from hypothyroid rats, FAT/CD36 was upregulated (mRNA levels and total tissue, sarcolemmal, and mitochondrial protein levels). Administration of either T3or T2to hypothyroid rats resulted in 1) little or no change in FAT/CD36 mRNA level, 2) a decreased total FAT/CD36 protein level, and 3) further increases in FAT/CD36 protein level in sarcolemma and mitochondria. Thus, the main effect of each iodothyronine seemed to be exerted at the level of FAT/CD36 cellular distribution. The effect of further increases in FAT/CD36 protein level in sarcolemma and mitochondria was already evident at 1 h after iodothyronine administration. Each iodothyronine increased the mitochondrial fatty acid oxidation rate. However, the mechanisms underlying their rapid effects seem to differ; T2and T3each induce FAT/CD36 translocation to mitochondria, but only T2induces increases in carnitine palmitoyl transferase system activity and in the mitochondrial substrate oxidation rate.

The worldwide prevalence of obesity-associated pathologies, including type 2 diabetes, requires thorough investigation of mechanisms and interventions. Recent studies have highlighted thyroid hormone analogs and derivatives as potential agents able to counteract such pathologies. In this study, in rats receiving a high-fat diet (HFD), we analyzed the effects of a 4-wk daily administration of a naturally occurring iodothyronine, 3,5-diiodo-L-thyronine (T2), on the gastrocnemius muscle metabolic/structural phenotype and insulin signaling. The HFD-induced increases in muscle levels of fatty acid translocase (3-fold; P

OBJECTIVE High-fat diets (HFDs) are known to induce insulin resistance. Previously, we showed that 3,5-diiodothyronine (T2), concomitantly administered to rats on a 4-week HFD, prevented gain in body weight and adipose mass. Here we investigated whether and how T2 prevented HFD-induced insulin resistance. RESEARCH DESIGN AND METHODS We investigated the biochemical targets of T2 related to lipid and glucose homeostasis over time using various techniques, including genomic and proteomic profiling, immunoblotting, transient transfection, and enzyme activity analysis. RESULTS Here we show that, in rats, HFD feeding induced insulin resistance (as expected), whereas T2 administration prevented its onset. T2 did so by rapidly stimulating hepatic fatty acid oxidation, decreasing hepatic triglyceride levels, and improving the serum lipid profile, while at the same time sparing skeletal muscle from fat accumulation. At the mechanistic level, 1) transfection studies show that T2 does not act via thyroid hormone receptor β; 2) AMP-activated protein kinase is not involved in triggering the effects of T2; 3) in HFD rats, T2 rapidly increases hepatic nuclear sirtuin 1 (SIRT1) activity; 4) in an in vitro assay, T2 directly activates SIRT1; and 5) the SIRT1 targets peroxisome proliferator–activated receptor (PPAR)-γ coactivator (PGC-1α) and sterol regulatory element–binding protein (SREBP)-1c are deacetylated with concomitant upregulation of genes involved in mitochondrial biogenesis and downregulation of lipogenic genes, and PPARα/δ-induced genes are upregulated, whereas genes involved in hepatic gluconeogenesis are downregulated. Proteomic analysis of the hepatic protein profile supported these changes. CONCLUSIONS T2, by activating SIRT1, triggers a cascade of events resulting in improvement of the serum lipid profile, prevention of fat accumulation, and, finally, prevention of diet-induced insulin resistance.

3,5-Diiodo-l-thyronine (T2) powerfully reduces adiposity in rats fed a high-fat diet (HFD), stimulating (in the liver) fatty acid oxidation and mitochondrial uncoupling, and strongly counteracting steatosis, a condition commonly associated with diet-induced obesity. Proteomics offer unique possibilities for the investigation of changes in the levels and modifications of proteins. Here, combining 2D-E, mass spectrometry, and blue native (BN) PAGE, we studied how the subcellular hepatic phenotype responds to HFD and T2-treatment. By identifying differentially expressed proteins and analyzing their interrelation [using the Ingenuity Pathway Analysis (IPA) platform], we obtained an integrated view of the phenotypic/metabolic adaptations occurring in the liver proteome during HFD with or without T2-treatment. Interestingly, T2 counteracted several HFD-induced changes, mostly in mitochondria. BN-PAGE and subsequent in-gel activity analysis of OXPHOS complexes revealed a modified profile of individual complexes in HFD mitochondria vs. normal ones. This pattern was re-normalized in mitochondria from T2-treated HFD animals. These data indicate that in HFD rats, the effects of T2 on the liver proteome cause it to resemble that associated with a non-steatotic condition. The identified metabolic pathways (mainly at the mitochondrial level) may be responsible for the beneficial effects of T2 on liver adiposity and metabolism. © 2010 The Royal Society of Chemistry.

Background/Aims: Mitochondrial dysfunction is central to the physiopathology of steatosis and/or non-alcoholic fatty liver disease. In this study on rats we investigated whether 3,5-diiodo-l-thyronine (T2), a biologically active iodothyronine, acting at mitochondrial level is able to reverse hepatic steatosis after its induction through a high-fat diet. Methods: Hepatic steatosis was induced by long-term high-fat feeding of rats for six weeks which were then fed the same high-fat diet for the next 4 weeks and were simultaneously treated or not treated with T2. Histological analyses were performed on liver sections (by staining with Sudan black B). In liver mitochondria fatty acid oxidation rate, mitochondrial efficiency (by measuring proton conductance) and mitochondrial oxidative stress (by measuring H2O2 release, aconitase and SOD activity) were detected. Results: Stained sections showed that T2 treatment reduced hepatic fatty accumulation induced by a high-fat diet. At the mitochondrial level, the fatty acid oxidation rate and carnitine palmitoyl transferase activity were enhanced by T2 treatment. Moreover, by stimulating mitochondrial uncoupling, T2 caused less efficient utilization of fatty acid substrates and ameliorated mitochondrial oxidative stress. Conclusion: These data demonstrate that T2, by activating mitochondrial processes, markedly reverses hepatic steatosis in vivo. © 2009 European Association for the Study of the Liver.

Hamster (Mesocricetus auratus) harderian gland (HG) is a dimorphic orbital gland producing a copious lipid secretion. Two cell-types are present in hamster HG, type I in both sexes, type II only in males. In hamster HGs, we found a marked sexual dichotomy in the expression of uncoupling protein-3 (UCP3), a mitochondrial protein carrier, that probably exports fatty acid anions and fatty acid peroxides from the mitochondrial matrix. Following castration and/or testosterone treatment: (1) UCP3 levels correlated with the type II-cell percentage, not with testosterone levels, (2) in male HGs, UCP3 was comparable to female levels at 30 days post-castration (when the type II-cell percentage had fallen from 50% to 5%), although testosterone was already near zero at 15 days (when neither the type II-cell percentage nor the UCP3 level had fallen), and testosterone-replacement therapy prevented these changes. Testosterone-treated females possessed type II cells and a UCP3 level about twofold higher than in control females. Males displayed more intense UCP3 immunohistochemical positivity in type I HG cells than females. Hence, testosterone may indirectly control UCP3 expression by regulating the gland’s morphological and lipid dimorphism. Straight-chain fatty acids [found in alkyl diacylglycerols (ADGs) in males] are oxidized predominantly in mitochondria, branched-chain fatty acids (abundant in ADGs in females) predominantly in peroxisomes, so we speculate that the higher UCP3 expression in males reflects greater fatty acid flux in HG mitochondria. This is supported by our finding that in female (not male) HGs, the peroxisome-rich fraction contained a-methylacyl-CoA racemase (AMACR), an enzyme important in the beta-oxidation of branched-chain fatty acids. Hamster (Mesocricetus auratus) harderian gland (HG) is a dimorphic orbital gland producing a copious lipid secretion. Two cell-types are present in hamster HG, type I in both sexes, type II only in males. In hamster HGs, we found a marked sexual dichotomy in the expression of uncoupling protein-3 (UCP3), a mitochondrial protein carrier, that probably exports fatty acid anions and fatty acid peroxides from the mitochondrial matrix. Following castration and/or testosterone treatment: (1) UCP3 levels correlated with the type II-cell percentage, not with testosterone levels, (2) in male HGs, UCP3 was comparable to female levels at 30 days post-castration (when the type II-cell percentage had fallen from 50% to 5%), although testosterone was already near zero at 15 days (when neither the type II-cell percentage nor the UCP3 level had fallen), and testosterone-replacement therapy prevented these changes. Testosterone-treated females possessed type II cells and a UCP3 level about twofold higher than in control females. Males displayed more intense UCP3 immunohistochemical positivity in type I HG cells than females. Hence, testosterone may indirectly control UCP3 expression by regulating the gland's morphological and lipid dimorphism. Straight-chain fatty acids [found in alkyl diacylglycerols (ADGs) in males] are oxidized predominantly in mitochondria, branched-chain fatty acids (abundant in ADGs in females) predominantly in peroxisomes, so we speculate that the higher UCP3 expression in males reflects greater fatty acid flux in HG mitochondria. This is supported by our finding that in female (not male) HGs, the peroxisome-rich fraction contained α-methylacyl-CoA racemase (AMACR), an enzyme important in the β-oxidation of branched-chain fatty acids. © 2008 Wiley-Liss, Inc.

3,5,3′-triiodo-L-thyronine (T3) modulates development and growth, and in adult life it exerts a profound effect on basal metabolic rate, increasing respiration rate and simultaneously lowering metabolic efficiency. It mainly acts through the coordinated and synergistic modulation of both nuclear and mitochondrial genome expressions giving rise to a complex network of factors and cellular events not yet completely defined. Thus, understanding the effects of T3 requires investigations at several levels [such as genes and gene transcripts (genome and transcriptome), proteins and metabolites, functions and metabolic assessment (proteome and metabolome)]. As yet, the ultimate effects of T3 on tissue-proteomes remain largely unknown. The proteins are excellent targets in disease diagnostics, prognostics, and therapeutics, and therefore proteomic approaches [such as two-dimensional gel electrophoresis (2D-E), two-dimensional liquid chromatography (2-DL), and mass spectrometry (MS)] represent powerful tools for a) the discovery of novel hormone/drug targets and biomarkers, and b) the study of in vivo and in vitro hormone effects on cellular metabolism and protein expressions. Increasingly proteomic techniques are being adopted, in particular to avoid the limitations inherent in the more classical approaches, to solve analytical problems and obtain a more comprehensive identification and characterization of molecular events associated with patho-physiological conditions. Here, we review the new leads emerging from the application of comparative proteomics to the actions of thyroid hormones, and we overview the technologies that can improve the resolution of proteins differing in hydrophobicity, intracellular location, complex formation, and membrane binding. ©2008 Bentham Science Publishers Ltd.

The processes and pathways mediating the intermediary metabolism of carbohydrates, lipids, and proteins are all affected by thyroid hormones (THs) in almost all tissues. Particular attention has been devoted by scientists to the effects of THs on lipid metabolism. Among others, effects related to cholesterol, lipid handling, and cardiac performance have been the subject of study. Many reports are present in the literature concerning the calorigenic effect of THs, with most of them aimed at identifying the molecular basis of this effect. However, at the moment the mechanism(s) underlying the metabolic effects of THs remain to be elucidated. THs exert most of their effects though TH receptors (TRs). However, some effects of THs cannot be explained by a nuclear-mediated pathway, and recently an increasing number of nonnuclear actions have been described, which can provide a regulatory system of which the effects differ from those mediated on the transcriptional level by TRs. Some of the TH derivatives (naturally occurring metabolites and analogs) possess biological activities. TH-related biological effects have been described for physiological products such as tetraiodothyroacetic acid (Tetrac) and triiodothyroacetic acid (Triac) (via oxidative deamination and decarboxylation of thyroxine [T4] and triiodothyronine [T3] alanine chain), 3,3′,5′- triiodothyronine (rT3) (via T4 and T3 deiodination), 3,3′-diiodothyronine (3,3′-T2) and 3,5-diiodothyronine (T2) (via T4, T3, and rT3 deiodination), and 3-iodothyronamine (T1AM) and thyronamine (T0AM) (via T4 and T3 deiodination and amino acid decarboxylation), as well as for TH structural analogs, such as 3,5,3′-triiodothyropropionic acid (Triprop), 3,5-dibromo-3-pyridazinone-l- thyronine (L-940901), N-[3,5-dimethyl-4-(4′-hydroxy-3′- isopropylphenoxy)-phenyl]-oxamic acid (CGS 23425), 3,5-dimethyl-4[(4′- hydroxy-3′-isopropylbenzyl)-phenoxy] acetic acid (GC-1), 3,5-dichloro-4[(4-hydroxy-3-isopropylphenoxy)phenyl] acetic acid (KB-141), and 3,5-diiodothyropropionic acid (DITPA). Most of these compounds have interesting properties: counteracting lipid accumulation, reducing cholesterol level, and increasing lipid metabolism without cardiotoxic effects. Hopefully, further studies on basic mechanisms of such compounds will be harbingers of more knowledge on the metabolic effects of TH derivatives and on their possible clinical application. © Copyright 2008, Mary Ann Liebert, Inc.

The peroxisome proliferator-activated receptors (PPARs), which are ligand-inducible transcription factors expressed in a variety of tissues, have been shown to perform key roles in lipid homeostasis. In physiological situations such as fasting and physical exercise, one PPAR subtype, PPARδ, triggers a transcriptional program in skeletal muscle leading to a switch in fuel usage from glucose/fatty acids to solely fatty acids, thereby drastically increasing its oxidative capacity. The metabolic action of PPARδ has also been verified in humans. In addition, it has become clear that the action of PPARδ is not restricted to skeletal muscle. Indeed, PPARδ has been shown to play a crucial role in whole-body lipid homeostasis as well as in insulin sensitivity, and it is active not only in skeletal muscle (as an activator of fat burning) but also in the liver (where it can activate glycolysis/lipogenesis, with the produced fat being oxidized in muscle) and in the adipose tissue (by incrementing lipolysis). The main aim of this review is to highlight the central role for activated PPARδ in the reversal of any tendency toward the development of insulin resistance. Copyright © 2008 Pieter de Lange et al.

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 proliferator-activated receptor (PPAR)gamma coactivator-1alpha (PGC-1alpha), PPARdelta, and their target genes, which are involved in the formation of oxidative muscle fibers, mitochondrial 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.

T(3) regulates energy metabolism by stimulating metabolic rate and decreasing metabolic efficiency. The discovery of mitochondrial uncoupling protein 3 (UCP3), its homology to UCP1, and regulation by T(3) rendered it a possible molecular determinant of the action of T(3) on energy metabolism, but data are controversial. This controversy may in part be attributable to discrepancies observed between the regulation by T(3) of UCP3 expression in rats, humans, and mice. To clarify this issue, we studied 1) the induction kinetics of the UCP3 gene by T(3) in rat skeletal muscle, 2) the influence of fatty acids, and 3) the structure and regulation of the various UCP3 promoters by T(3). Within 8 h of single-dose T(3) administration, hypothyroid rats showed a rise in serum fatty acid levels concomitant with a rapid increase in UCP3 expression in gastrocnemius muscle, followed by inductions of peroxisome proliferator activated receptor delta (PPARdelta) (within 24 h) and PPAR target gene expression (after 24 h). This T(3)-induced early UCP3 expression depended on fatty acid-PPAR signaling because depleting serum fatty acid levels abolished its expression, restorable by administration of the PPARdelta agonist L165,041 (4-[3-(4-acetyl-3-hydroxy-2-propylphenoxy)propoxy]phenoxy]acetic acid). In transfected rat L6 myoblasts, only the rat UCP3 promoter positively responded to T(3) and L165,041 together in the presence of MyoD, thyroid hormone receptor beta1 (TRbeta1), PPARdelta, or PPARdelta plus the TR dimerization partner retinoid X receptor alpha. All promoters share a response element common to TR and PPAR (TRE 1), but the observed species differences may be attributable to different localizations of the MyoD response element, which in the rat maps to exon 1.

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, appoximately 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.

We investigated the role of uncoupling protein 3 (UCP3) during fasting and examined the effect of triiodothyronine (T3) administration in such a condition. The possible involvement of mitochondrial thioesterase (MTE I) and the role of putative cofactors, such as coenzyme Q (CoQ), was also examined. Here, we report that fasting induced a more than twofold elevation in the expression and activity of MTE I, and an increase in UCP3 expression, without any associated uncoupling activity. Administration of T3 to fasting rats further up-regulated UCP3 as well as MTE I expression, markedly enhanced MTE I enzyme activity and prevented the impairment of the uncoupling activity of UCP3 normally seen during fasting. Indeed, T3-treatment induced an UCP3-dependent decrease in mitochondrial membrane potential, which was abolished by the addition of either GDP or superoxide dismutase (SOD). T3 administration also prevented the marked decrease of CoQ levels observed in fasting rats and this provides evidence that also, in vivo, CoQ represents an essential cofactor for the UCP3-mediated uncoupling. The data also show that MTE I and UCP3 are likely involved in the same biochemical mechanism and that UCP3 postulated functions, such as lipid handling and uncoupling, are not mutually exclusive but may coexist in vivo.

Thyroid hormones increase energy expenditure, partly by reducing metabolic efficiency. The control of specific genes at the transcriptional level is thought to be the major molecular mechanism. However, both the number and the identity of the thyroid hormone-controlled genes remain unknown, as do their relative contributions. Uncoupling protein-3, a recently identified member of the mitochondrial transporter superfamily and one that is predominantly expressed in skeletal muscle, has the potential to be a molecular determinant for thyroid thermogenesis. However, changes in mitochondrial proton conductance and resting metabolic rate after physiologically mediated changes in uncoupling protein-3 levels have not been described. Here, in a study on hypothyroid rats given a single injection of T(3), we describe a strict correlation in terms of time course between the induced increase in uncoupling protein-3 expression (at mRNA and protein levels) and decrease in mitochondrial respiratory efficiency, on the one hand, and the increase in resting metabolic rate, on the other. First, we describe our finding that uncoupling protein-3 is present and regulated by T(3) only in metabolically relevant tissues (such as skeletal muscle and heart). Second, we follow the time course (at 0, 6, 12, 24, 48, 65, 96, and 144 h) of both uncoupling protein-3 mRNA levels and mitochondrial uncoupling protein-3 density in gastrocnemius muscle and heart. In both tissues, the maximal (12-fold) increase in uncoupling protein-3 density was reached at 65 h. The resting metabolic rate [lO(2)(kg(0.75))(-1)h(-1)] showed the same time course, and at 65 h the increase vs. time zero was 45% (1.316 +/- 0.026 vs. 0.940 +/- 0.007; P < 0.001). At the same time point, gastrocnemius muscle mitochondria showed a significantly higher nonphosphorylating respiration rate (nanoatoms of oxygen per min/mg protein; increase vs. time zero, 40%; 118 +/- 4 vs. 85 +/- 9; P < 0.05), whereas the membrane potential decreased by 8% (168 +/- 2 vs. 182 +/- 4; P < 0.05). These data are diagnostic of mitochondrial uncoupling. The results reported here provide the first direct in vivo evidence that uncoupling protein-3 has the potential to act as a molecular determinant in the regulation of resting metabolic rate by T(3).

Cortisol resistance (CR) is a rare disease characterized by a generalized reduced sensitivity of end-organs to the actions of glucocorticoids (GCs). GC effects are mediated by the GC receptor (GR). The molecular alterations in CR described thus far were located in the hormone-binding domain of the GR gene. Recent reports of a considerable prevalence of abnormalities in the GR in patients attending the endocrine clinic prompted us to carry out further investigations with respect to GR protein and GR gene in patients attending the endocrine clinic for a broad spectrum of complaints and biochemical evidence suggesting a CR. In the present study, we describe five patients with biochemical and clinical CR. All patients showed a diurnal rhythm of serum cortisol concentrations (albeit at a high level), an insufficient suppression of serum cortisol concentration in reaction to 1 mg dexamethasone (DEX), and variable degrees of androgen overproduction, in the absence of clinical signs and symptoms of Cushing’s syndrome. Three of the four female patients presented with complaints of androgen overproduction, two of them in combination with fatigue. The other female patient had severe steroid-resistant asthma. The only male patient and his son were asymptomatic. In four patients, we investigated receptor protein characteristics on mononuclear leukocytes in a whole cell DEX binding assay and studied the ability of DEX to inhibit mitogen-induced cell proliferation in mononuclear leukocytes in vitro. In all patients investigated, we found alterations in receptor number or ligand affinity and/or the ability of DEX to inhibit mitogen-induced cell proliferation. To investigate the molecular defects leading to the clinical and biochemical pictures in these patients, we screened the GR gene using PCR/single-strand conformational polymorphism/sequence analysis. No GR gene alterations were found in these patients. In conclusion, the five patients described had clinical and biochemical evidence of CR, but no abnormalities were demonstrated in the GR gene. Probably, as yet undefined alterations somewhere in the cascade of events starting with ligand binding to the GR protein, and finally resulting in the regulation of the expression of GC responsive genes, or postreceptor defects or interactions with other nuclear factors form the pathophysiologic basis of CR in these patients.

The beta isoform of the human glucocorticoid receptor, hGRbeta, is a product of alternative splicing of the hGR gene. The physiological function of this isoform is unknown up to now. Recent data are contradictory in that they either favor or argue against a role of hGRbeta as a repressor of the functional hGRalpha isoform. In the present study hGRbeta did not inhibit transcriptional activation of the MMTV-driven luciferase reporter gene by dexamethasone-activated hGRalpha in COS-1 cells. In addition, two naturally occurring variants of the hGRbeta isoform associated with altered sensitivity to glucocorticoids, termed hGRbeta-R23K and hGRbeta-N363S, did not repress hGRalpha, even when overexpressed 10-fold. We conclude that the hGRbeta isoform, as well as two of its natural variants, do not act as dominant negative inhibitors of hGRalpha function and that the beta isoform does not appear to play a role in the regulation of glucocorticoid sensitivity.

Using differentiated rat L6 cells, we studied the direct effect of 3,5,3'-triiodo-l-thyronine (T3) and 3,5-diiodo-l-thyronine (T2) on the response to insulin in presence of fatty acids with a varying degree of saturation. We found that T3 and T2 both invert the response to insulin by modulating Akt Ser473 phosphorylation in the presence of palmitate and oleate. Both hormones prevented palmitate-induced insulin resistance, whereas increased insulin sensitivity in the presence of oleate was reduced, with normalization to (or, in the case of T3, even below) control levels. Both hormones effectively reduced intracellular acylcarnitine concentrations. Interestingly, insulin sensitization was lowered by incubation of the myotubes with relevant concentrations of palmitoylcarnitines (C16) and increased by oleylcarnitines and linoleylcarnitines (C18:1 and C18:2, respectively). The efficiency of mitochondrial respiration decreased in the order palmitate-oleate-linoleate; in the presence of palmitate, only T3 increased ATP synthesis-independent cellular respiration and mitochondrial respiratory complex activities. Both hormones modulated gene expression and enzyme activities related to insulin sensitivity, glucose metabolism, and lipid handling. Although T2 and T3 differentially regulated the expression of relevant genes involved in glucose metabolism, they equally stimulated related metabolic activities. T2 and T3 differentially modulated mitochondrial fatty acid uptake and oxidation in the presence of each fatty acid. The results show that T2 and T3 both invert the fatty acid-induced response to insulin but through different mechanisms, and that the outcome depends on the degree of saturation of the fatty acids and their derived acylcarnitines.-Giacco, A., delli Paoli, G., Senese, R., Cioffi, F., Silvestri, E., Moreno, M., Ruoppolo, M., Caterino, M., Costanzo, M., Lombardi, A., Goglia, F., Lanni, A., de Lange, P. The saturation degree of fatty acids and their derived acylcarnitines determines the direct effect of metabolically active thyroid hormones on insulin sensitivity in skeletal muscle cells.