Ablation of uncoupling protein 3 affects interrelated factors leading to lipolysis and insulin resistance in visceral white adipose tissue.

The physiological role played by uncoupling protein 3 (UCP3) in white adipose tissue (WAT) has not been elucidated so far. In the present study, we evaluated the impact of the absence of the whole body UCP3 on WAT physiology in terms of ability to store triglycerides, oxidative capacity, response to insulin, inflammation, and adipokine production. Wild type (WT) and UCP3 Knockout (KO) mice housed at thermoneutrality (30°C) have been used as the animal model. Visceral gonadic WAT (gWAT) from KO mice showed an impaired capacity to store triglycerides (TG) as indicated by its lowered weight, reduced adipocyte diameter, and higher glycerol release (index of lipolysis). The absence of UCP3 reduces the maximal oxidative capacity of gWAT, increases mitochondrial free radicals, and activates ER stress. These processes are associated with increased levels of monocyte chemoattractant protein-1 and TNF-α. The response of gWAT to in vivo insulin administration, revealed by (ser473)-AKT phosphorylation, was blunted in KO mice, with a putative role played by eif2a, JNK, and inflammation. Variations in adipokine levels in the absence of UCP3 were observed, including reduced adiponectin levels both in gWAT and serum. As a whole, these data indicate an important role of UCP3 in regulating the metabolic functionality of gWAT, with its absence leading to metabolic derangement. The obtained results help to clarify some aspects of the association between metabolic disorders and low UCP3 levels.

Physiological Approaches Targeting Cellular and Mitochondrial Pathways Underlying Adipose Organ Senescence.

The adipose organ is involved in many metabolic functions, ranging from the production of endocrine factors to the regulation of thermogenic processes. Aging is a natural process that affects the physiology of the adipose organ, leading to metabolic disorders, thus strongly impacting healthy aging. Cellular senescence modifies many functional aspects of adipose tissue, leading to metabolic alterations through defective adipogenesis, inflammation, and aberrant adipocytokine production, and in turn, it triggers systemic inflammation and senescence, as well as insulin resistance in metabolically active tissues, leading to premature declined physiological features. In the various aging fat depots, senescence involves a multiplicity of cell types, including mature adipocytes and immune, endothelial, and progenitor cells that are aging, highlighting their involvement in the loss of metabolic flexibility, one of the common features of aging-related metabolic disorders. Since mitochondrial stress represents a key trigger of cellular senescence, and senescence leads to the accumulation of abnormal mitochondria with impaired dynamics and hindered homeostasis, this review focuses on the beneficial potential of targeting mitochondria, so that strategies can be developed to manage adipose tissue senescence for the treatment of age-related metabolic disorders.

Absence of uncoupling protein 3 at thermoneutrality influences brown adipose tissue mitochondrial functionality in mice.

The physiological role played by uncoupling protein 3 (UCP3) in brown adipose tissue (BAT) has not been fully elucidated so far. In the present study, we evaluated the impact of the absence of UCP3 on BAT mitochondrial functionality and morphology. To this purpose, wild type (WT) and UCP3 Knockout (KO) female mice were housed at thermoneutrality (30°C), a condition in which BAT contributes to energy homeostasis independently of its cold-induced thermogenic function. BAT mitochondria from UCP3 KO mice presented a lower ability to oxidize the fatty acids and glycerol-3-phosphate, and an enhanced oxidative stress as revealed by enhanced mitochondrial electron leak, lipid hydroperoxide levels, and induction of antioxidant mitochondrial enzymatic capacity. The absence of UCP3 also influenced the mitochondrial super-molecular protein aggregation, an important feature for fatty acid oxidation rate as well as for adequate cristae organization and mitochondrial shape. Indeed, electron microscopy revealed alterations in mitochondrial morphology in brown adipocytes from KO mice. In the whole, data here reported show that the absence of UCP3 results in a significant alteration of BAT mitochondrial physiology and morphology. These observations could also help to clarify some aspects of the association between metabolic disorders associated with low UCP3 levels, as previously reported in human studies.

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.

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.

Short-Term, Combined Fasting and Exercise Improves Body Composition in Healthy Males.

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 < .001) and fat mass (-3.3 ± 1.3 kg; p < .001). Average lean mass was lost (-0.6 ± 1.5 kg; p < .001), but lean mass as a percentage of total body mass was not reduced. The authors observed a loss of -3.9 ± 1.9% android fat over total fat mass (p < .001), a loss of -2.2 ± 1.9% gynoid over total fat mass (p < .001), and reduced android/gynoid ratios (-0.05 ± 0.1; p < .001). Analyzing 15 preselected single-nucleotide polymorphisms in 13 metabolism-related genes revealed trending associations for thyroid state-related single-nucleotide polymorphisms rs225014 (deiodinase 2) and rs35767 (insulin-like growth factor1), and rs1053049 (PPARD). In conclusion, a short period of combined fasting and exercise leads to a substantial loss of body and fat mass without a loss of lean mass as a percentage of total mass.

Differential Effects of 3,5-Diiodo-L-Thyronine and 3,5,3'-Triiodo-L-Thyronine On Mitochondrial Respiratory Pathways in Liver from Hypothyroid Rats.

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.

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 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.

Comparative effects of 3,5-diiodo-L-thyronine and 3,5,3'-triiodo-L-thyronine on mitochondrial damage and cGAS/STING-driven inflammation in liver of hypothyroid rats.

Maintaining a well-functioning mitochondrial network through the mitochondria quality control (MQC) mechanisms, including biogenesis, dynamics and mitophagy, is crucial for overall health. Mitochondrial dysfunction caused by oxidative stress and further exacerbated by impaired quality control can trigger inflammation through the release of the damage-associated molecular patterns (mtDAMPs). mtDAMPs act by stimulating the cyclic GMP-AMP synthase (cGAS) stimulator of interferon genes (STING) pathway. Recently, aberrant signalling of the cGAS-STING axis has been recognised to be closely associated with several sterile inflammatory diseases (e.g. non-alcoholic fatty liver disease, obesity). This may fit the pathophysiology of hypothyroidism, an endocrine disorder characterised by the reduction of thyroid hormone production associated with impaired metabolic fluxes, oxidative balance and inflammatory status. Both 3,5,3'-triiodo-L-tyronine (T3) and its derivative 3,5-diiodo-L-thyronine (3,5-T2), are known to mitigate processes targeting mitochondria, albeit the underlying mechanisms are not yet fully understood. Therefore, we used a chemically induced hypothyroidism rat model to investigate the effect of 3,5-T2 or T3 administration on inflammation-related factors (inflammatory cytokines, hepatic cGAS-STING pathway), oxidative stress, antioxidant defence enzymes, mitochondrial DNA (mtDNA) damage, release and repair, and the MQC system in the liver. Hypothyroid rats showed: i) increased oxidative stress, ii) accumulation of mtDNA damage, iii) high levels of circulating cytokines, iv) hepatic activation of cGAS-STING pathways and v) impairment of MQC mechanisms and autophagy. Both iodothyronines restored oxidative balance by enhancing antioxidant defence, preventing mtDNA damage through the activation of mtDNA repair mechanisms (OGG1, APE1, and POLγ) and promoting autophagy progression. Concerning MQC, both iodothyronines stimulated mitophagy and dynamics, with 3,5-T2 activating fusion and T3 modulating both fusion and fission processes. Moreover, only T3 enhanced mitochondrial biogenesis. Notably, 3,5-T2, but not T3, reversed the hypothyroidism-induced activation of the cGAS-STING inflammatory cascade. In addition, it is noteworthy that 3,5-T2 seems more effective than T3 in reducing circulating pro-inflammatory cytokines IL-6 and IL-1B and in stimulating the release of IL-10, a known anti-inflammatory cytokine. These findings reveal novel molecular mechanisms of hepatic signalling pathways involved in hypothyroidism, which could be targeted by natural iodothyronines, particularly 3,5-T2, paving the way for the development of new treatment strategies for inflammatory diseases.

Effect of CB1 Receptor Deficiency on Mitochondrial Quality Control Pathways in Gastrocnemius Muscle.

This study aims to explore the complex role of cannabinoid type 1 receptor (CB1) signaling in the gastrocnemius muscle, assessing physiological processes in both CB1+/+ and CB1-/- mice. The primary focus is to enhance our understanding of how CB1 contributes to mitochondrial homeostasis. At the tissue level, CB1-/- mice exhibit a substantial miRNA-related alteration in muscle fiber composition, characterized by an enrichment of oxidative fibers. CB1 absence induces a significant increase in the oxidative capacity of muscle, supported by elevated in-gel activity of Complex I and Complex IV of the mitochondrial respiratory chain. The increased oxidative capacity is associated with elevated oxidative stress and impaired antioxidant defense systems. Analysis of mitochondrial biogenesis markers indicates an enhanced capacity for new mitochondria production in CB1-/- mice, possibly adapting to altered muscle fiber composition. Changes in mitochondrial dynamics, mitophagy response, and unfolded protein response (UPR) pathways reveal a dynamic interplay in response to CB1 absence. The interconnected mitochondrial network, influenced by increased fusion and mitochondrial UPR components, underlines the dual role of CB1 in regulating both protein quality control and the generation of new mitochondria. These findings deepen our comprehension of the CB1 impact on muscle physiology, oxidative stress, and MQC processes, highlighting cellular adaptability to CB1-/-. This study paves the way for further exploration of intricate signaling cascades and cross-talk between cellular compartments in the context of CB1 and mitochondrial homeostasis.

(Healthy) ageing: focus on iodothyronines.

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.

Adipose Tissue Remodeling in Obesity: An Overview of the Actions of Thyroid Hormones and Their Derivatives.

Metabolic syndrome and obesity have become important health issues of epidemic proportions and are often the cause of related pathologies such as type 2 diabetes (T2DM), hypertension, and cardiovascular disease. Adipose tissues (ATs) are dynamic tissues that play crucial physiological roles in maintaining health and homeostasis. An ample body of evidence indicates that in some pathophysiological conditions, the aberrant remodeling of adipose tissue may provoke dysregulation in the production of various adipocytokines and metabolites, thus leading to disorders in metabolic organs. Thyroid hormones (THs) and some of their derivatives, such as 3,5-diiodo-l-thyronine (T2), exert numerous functions in a variety of tissues, including adipose tissues. It is known that they can improve serum lipid profiles and reduce fat accumulation. The thyroid hormone acts on the brown and/or white adipose tissues to induce uncoupled respiration through the induction of the uncoupling protein 1 (UCP1) to generate heat. Multitudinous investigations suggest that 3,3',5-triiodothyronine (T3) induces the recruitment of brown adipocytes in white adipose depots, causing the activation of a process known as "browning". Moreover, in vivo studies on adipose tissues show that T2, in addition to activating brown adipose tissue (BAT) thermogenesis, may further promote the browning of white adipose tissue (WAT), and affect adipocyte morphology, tissue vascularization, and the adipose inflammatory state in rats receiving a high-fat diet (HFD). In this review, we summarize the mechanism by which THs and thyroid hormone derivatives mediate adipose tissue activity and remodeling, thus providing noteworthy perspectives on their efficacy as therapeutic agents to counteract such morbidities as obesity, hypercholesterolemia, hypertriglyceridemia, and insulin resistance.

Exercise Equals the Mobilization of Visceral versus Subcutaneous Adipose Fatty Acid Molecules in Fasted Rats Associated with the Modulation of the AMPK/ATGL/HSL Axis.

Combining exercise with fasting is known to boost fat mass-loss, but detailed analysis on the consequential mobilization of visceral and subcutaneous WAT-derived fatty acids has not been performed. In this study, a subset of fasted male rats (66 h) was submitted to daily bouts of mild exercise. Subsequently, by using gas chromatography-flame ionization detection, the content of 22 fatty acids (FA) in visceral (v) versus subcutaneous (sc) white adipose tissue (WAT) depots was compared to those found in response to the separate events. Findings were related to those obtained in serum and liver samples, the latter taking up FA to increase gluconeogenesis and ketogenesis. Each separate intervention reduced scWAT FA content, associated with increased levels of adipose triglyceride lipase (ATGL) protein despite unaltered AMP-activated protein kinase (AMPK) Thr172 phosphorylation, known to induce ATGL expression. The mobility of FAs from vWAT during fasting was absent with the exception of the MUFA 16:1 n-7 and only induced by combining fasting with exercise which was accompanied with reduced hormone sensitive lipase (HSL) Ser563 and increased Ser565 phosphorylation, whereas ATGL protein levels were elevated during fasting in association with the persistently increased phosphorylation of AMPK at Thr172 both during fasting and in response to the combined intervention. As expected, liver FA content increased during fasting, and was not further affected by exercise, despite additional FA release from vWAT in this condition, underlining increased hepatic FA metabolism. Both fasting and its combination with exercise showed preferential hepatic metabolism of the prominent saturated FAs C:16 and C:18 compared to the unsaturated FAs 18:1 n-9 and 18:2 n-6:1. In conclusion, depot-specific differences in WAT fatty acid molecule release during fasting, irrelevant to their degree of saturation or chain length, are mitigated when combined with exercise, to provide fuel to surrounding organs such as the liver which is correlated with increased ATGL/ HSL ratios, involving AMPK only in vWAT.

Responses of skeletal muscle lipid metabolism in rat gastrocnemius to hypothyroidism and iodothyronine administration: a putative role for FAT/CD36.

Iodothyronines such as triiodothyronine (T(3)) and 3,5-diiodothyronine (T(2)) 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 T(3) and T(2) on 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 T(3) or T(2) administration 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 T(3) or T(2) to 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; T(2) and T(3) each induce FAT/CD36 translocation to mitochondria, but only T(2) induces increases in carnitine palmitoyl transferase system activity and in the mitochondrial substrate oxidation rate.

3,5-Diiodo-L-thyronine prevents high-fat-diet-induced insulin resistance in rat skeletal muscle through metabolic and structural adaptations.

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<0.05) and TGs (2-fold, P<0.05) were prevented by T2 (each; P<0.05 vs. HFD). T2 increased insulin-stimulated Akt phosphorylation levels (∼2.5-fold; P<0.05 vs. HFD). T2 induced these effects while sparing muscle mass and without cardiac hypertrophy. T2 increased the muscle contents of fast/glycolytic fibers (2-fold; P<0.05 vs. HFD) and sarcolemmal glucose transporter 4 (3-fold; P<0.05 vs. HFD). Adipocyte differentiation-related protein was predominantly present within the slow/oxidative fibers in HFD-T2. In T2-treated rats (vs. HFD), glycolytic enzymes and associated components were up-regulated (proteomic analysis, significance limit: 2-fold; P<0.05), as was phosphofructokinase activity (by 1.3-fold; P<0.05), supporting the metabolic shift toward a more glycolytic phenotype. These results highlight T2 as a potential therapeutic approach to the treatment of diet-induced metabolic dysfunctions.

Mild Endurance Exercise during Fasting Increases Gastrocnemius Muscle and Prefrontal Cortex Thyroid Hormone Levels through Differential BHB and BCAA-Mediated BDNF-mTOR Signaling in Rats.

Mild endurance exercise has been shown to compensate for declined muscle quality and may positively affect the brain under conditions of energy restriction. Whether this involves brain-derived neurotrophic factor (BDNF) and mammalian target of rapamycin (mTOR) activation in relation to central and peripheral tissue levels of associated factors such as beta hydroxy butyrate (BHB), branched-chain amino acids (BCAA) and thyroid hormone (T3) has not been studied. Thus, a subset of male Wistar rats housed at thermoneutrality that were fed or fasted was submitted to 30-min-mild treadmill exercise bouts (five in total, twice daily, 15 m/min, 0° inclination) over a period of 66 h. Prefrontal cortex and gastrocnemius muscle BHB, BCAA, and thyroid hormone were measured by LC-MS/MS analysis and were related to BDNF and mammalian target of rapamycin (mTOR) signaling. In gastrocnemius muscle, mild endurance exercise during fasting maintained the fasting-induced elevated BHB levels and BDNF-CREB activity and unlocked the downstream Akt-mTORC1 pathway associated with increased tissue BCAA. Consequently, deiodinase 3 mRNA levels decreased whereas increased phosphorylation of the mTORC2 target FOXO1 was associated with increased deiodinase 2 mRNA levels, accounting for the increased T3 tissue levels. These events were related to increased expression of CREB and T3 target genes beneficial for muscle quality previously observed in this condition. In rat L6 myoblasts, BHB directly induced BDNF transcription and maturation. Mild endurance exercise during fasting did not increase prefrontal cortex BHB levels nor was BDNF activated, whereas increased leucine levels were associated with Akt-independent increased phosphorylation of the mTORC1 target P70S6K. The associated increased T3 levels modulated the expression of known T3-target genes involved in brain tissue maintenance. Our observation that mild endurance exercise modulates BDNF, mTOR and T3 during fasting provides molecular clues to explain the observed beneficial effects of mild endurance exercise in settings of energy restriction.

Altered Mitochondrial Quality Control in Rats with Metabolic Dysfunction-Associated Fatty Liver Disease (MAFLD) Induced by High-Fat Feeding.

Metabolic dysfunction-associated fatty liver disease (MAFLD) is defined as the presence of hepatic steatosis in addition to one of three metabolic conditions: overweight/obesity, type 2 diabetes mellitus, or metabolic dysregulation. Chronic exposure to excess dietary fatty acids may cause hepatic steatosis and metabolic disturbances. The alteration of the quality of mitochondria is one of the factors that could contribute to the metabolic dysregulation of MAFDL. This study was designed to determine, in a rodent model of MAFLD, the effects of a long-term high-fat diet (HFD) on some hepatic processes that characterize mitochondrial quality control, such as biogenesis, dynamics, and mitophagy. To mimic the human manifestation of MAFLD, the rats were exposed to both an HFD and a housing temperature within the rat thermoneutral zone (28-30 °C). After 14 weeks of the HFD, the rats showed significant fat deposition and liver steatosis. Concomitantly, some important factors related to the hepatic mitochondrial quality were markedly affected, such as increased mitochondrial reactive oxygen species (ROS) production and mitochondrial DNA (mtDNA) damage; reduced mitochondrial biogenesis, mtDNA copy numbers, mtDNA repair, and mitochondrial fusion. HFD-fed rats also showed an impaired mitophagy. Overall, the obtained data shed new light on the network of different processes contributing to the failure of mitochondrial quality control as a central event for mitochondrial dysregulation in MAFLD.

UCP3 translocates lipid hydroperoxide and mediates lipid hydroperoxide-dependent mitochondrial uncoupling.

Although the literature contains many studies on the function of UCP3, its role is still being debated. It has been hypothesized that UCP3 may mediate lipid hydroperoxide (LOOH) translocation across the mitochondrial inner membrane (MIM), thus protecting the mitochondrial matrix from this very aggressive molecule. However, no experiments on mitochondria have provided evidence in support of this hypothesis. Here, using mitochondria isolated from UCP3-null mice and their wild-type littermates, we demonstrate the following. (i) In the absence of free fatty acids, proton conductance did not differ between wild-type and UCP3-null mitochondria. Addition of arachidonic acid (AA) to such mitochondria induced an increase in proton conductance, with wild-type mitochondria showing greater enhancement. In wild-type mitochondria, the uncoupling effect of AA was significantly reduced both when the release of O2* in the matrix was inhibited and when the formation of LOOH was inhibited. In UCP3-null mitochondria, however, the uncoupling effect of AA was independent of the above mechanisms. (ii) In the presence of AA, wild-type mitochondria released significantly more LOOH compared with UCP3-null mitochondria. This difference was abolished both when UCP3 was inhibited by GDP and under a condition in which there was reduced LOOH formation on the matrix side of the MIM. These data demonstrate that UCP3 is involved both in mediating the translocation of LOOH across the MIM and in LOOH-dependent mitochondrial uncoupling.

Uncoupling protein 3 expression levels influence insulin sensitivity, fatty acid oxidation, and related signaling pathways.

Controversy exists on whether uncoupling protein 3 (UCP3) positively or negatively influences insulin sensitivity in vivo, and the underlying signaling pathways have been scarcely studied. We studied how a progressive reduction in UCP3 expression (using UCP3 +/+, UCP3 +/-, and UCP3 -/- mice) modulates insulin sensitivity and related metabolic parameters. In order to further validate our observations, we also studied animals in which insulin resistance was induced by administration of a high-fat diet (HFD). In UCP3 +/- and UCP3 -/- mice, gastrocnemius muscle Akt/protein kinase B (Akt/PKB) (serine 473) and AMP-activated protein kinase (AMPK) (threonine 171) phosphorylation, and glucose transporter 4 (GLUT4) membrane levels were reduced compared to UCP3 +/+ mice. The HOMA-IR index (insulin resistance parameter) was increased both in the UCP3 +/- and UCP3 -/- mice. In these mice, insulin administration normalized Akt/PKB phosphorylation between genotypes while AMPK phosphorylation was further reduced, and sarcolemmal GLUT4 levels were induced but did not reach control levels. Furthermore, non-insulin-stimulated muscle fatty acid oxidation and the expression of several involved genes both in muscle and in liver were reduced. HFD administration induced insulin resistance in UCP3 +/+ mice and the aforementioned parameters resulted similar to those of chow-fed UCP3 +/- and UCP3 -/- mice. In conclusion, high-fat-diet-induced insulin resistance in wild-type mice mimics that of chow-fed UCP3 +/- and UCP3 -/- mice showing that progressive reduction of UCP3 levels results in insulin resistance. This is accompanied by decreased fatty acid oxidation and a less intense Akt/PKB and AMPK signaling.

TRC150094, a novel functional analog of iodothyronines, reduces adiposity by increasing energy expenditure and fatty acid oxidation in rats receiving a high-fat diet.

Chronic overnutrition and modern lifestyles 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<0.05) and body weight (BW) gain comparable to that of standard chow-fed (N) rats [N, HFD, and HFD-TRC rats, +97 g, +140 g (P<0.05 vs. N), and +98 g (P<0.05 vs. HFD)]. HFD-TRC rats had significantly less visceral adipose tissue (vs. HFD rats) and exhibited altered metabolism in two major tissues that are very active metabolically. In liver, mitochondrial fatty acid import and oxidation were increased (+56 and +32%, respectively; P<0.05 vs. HFD rats), and consequently the hepatic triglyceride content was lower (-35%; P<0.05 vs. HFD rats). These effects were independent of the AMP-activated protein kinase-acetyl CoA-carboxylase-malonyl CoA pathway but involved sirtuin 1 activation. In skeletal muscle, TRC induced a fiber shift toward the oxidative type in tibialis anterior muscle, increasing its capacity to oxidize fatty acids. HFD-TRC rats had lower (vs. HFD rats) plasma cholesterol and triglyceride concentrations. If reproduced in humans, these results will open interesting possibilities regarding the counteraction of metabolic dysfunction associated with ectopic/visceral fat accumulation.

Studies of complex biological systems with applications to molecular medicine: the need to integrate transcriptomic and proteomic approaches.

Omics approaches to the study of complex biological systems with potential applications to molecular medicine are attracting great interest in clinical as well as in basic biological research. Genomics, transcriptomics and proteomics are characterized by the lack of an a priori definition of scope, and this gives sufficient leeway for investigators (a) to discern all at once a globally altered pattern of gene/protein expression and (b) to examine the complex interactions that regulate entire biological processes. Two popular platforms in "omics" are DNA microarrays, which measure messenger RNA transcript levels, and proteomic analyses, which identify and quantify proteins. Because of their intrinsic strengths and weaknesses, no single approach can fully unravel the complexities of fundamental biological events. However, an appropriate combination of different tools could lead to integrative analyses that would furnish new insights not accessible through one-dimensional datasets. In this review, we will outline some of the challenges associated with integrative analyses relating to the changes in metabolic pathways that occur in complex pathophysiological conditions (viz. ageing and altered thyroid state) in relevant metabolically active tissues. In addition, we discuss several new applications of proteomic analysis to the investigation of mitochondrial activity.