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.

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.

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.

Adaptive Thermogenesis Driving Catch-Up Fat Is Associated With Increased Muscle Type 3 and Decreased Hepatic Type 1 Iodothyronine Deiodinase Activities: A Functional and Proteomic Study.

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.

Absence of Uncoupling Protein-3 at Thermoneutrality Impacts Lipid Handling and Energy Homeostasis in Mice.

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 °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.

Environmental Pollutants Effect on Brown Adipose Tissue.

Brown adipose tissue (BAT) with its thermogenic function due to the presence of the mitochondrial uncoupling protein 1 (UCP1), has been positively associated with improved resistance to obesity and metabolic diseases. During recent years, the potential influence of environmental pollutants on energetic homoeostasis and obesity development has drawn increased attention. The purpose of this review is to discuss how regulation of BAT function could be involved in the environmental pollutant effect on body energy metabolism. We mainly focused in reviewing studies on animal models, which provide a better insight into the cellular mechanisms involved in this effect on body energy metabolism. The current literature supports the hypothesis that some environmental pollutants, acting as endocrine disruptors (EDCs), such as dichlorodiphenyltrichoroethane (DDT) and its metabolite dichlorodiphenylethylene (DDE) as well as some, traffic pollutants, are associated with increased obesity risk, whereas some other chemicals, such as perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), had a reverse association with obesity. Noteworthy, the EDCs associated with obesity and metabolic disorders impaired BAT mass and function. Perinatal exposure to DDT impaired BAT thermogenesis and substrate utilization, increasing susceptibility to metabolic syndrome. Ambient particulate air pollutions induced insulin resistance associated with BAT mitochondrial dysfunction. On the other hand, the environmental pollutants (PFOS/PFOA) elicited a reduction in body weight and adipose mass associated with upregulation of UCP1 and increased oxidative capacity in brown-fat mitochondria. Further research is needed to better understand the physiological role of BAT in response to exposure to both obesogenic and anti-obesogenic pollutants and to confirm the same role in humans.

Correction: Akap1 Deficiency Promotes Mitochondrial Aberrations and Exacerbates Cardiac Injury Following Permanent Coronary Ligation via Enhanced Mitophagy and Apoptosis.

[This corrects the article DOI: 10.1371/journal.pone.0154076.].

Akap1 Deficiency Promotes Mitochondrial Aberrations and Exacerbates Cardiac Injury Following Permanent Coronary Ligation via Enhanced Mitophagy and Apoptosis.

A-kinase anchoring proteins (AKAPs) transmit signals cues from seven-transmembrane receptors to specific sub-cellular locations. Mitochondrial AKAPs encoded by the Akap1 gene have been shown to modulate mitochondrial function and reactive oxygen species (ROS) production in the heart. Under conditions of hypoxia, mitochondrial AKAP121 undergoes proteolytic degradation mediated, at least in part, by the E3 ubiquitin ligase Seven In-Absentia Homolog 2 (Siah2). In the present study we hypothesized that Akap1 might be crucial to preserve mitochondrial function and structure, and cardiac responses to myocardial ischemia. To test this, eight-week-old Akap1 knockout mice (Akap1-/-), Siah2 knockout mice (Siah2-/-) or their wild-type (wt) littermates underwent myocardial infarction (MI) by permanent left coronary artery ligation. Age and gender matched mice of either genotype underwent a left thoracotomy without coronary ligation and were used as controls (sham). Twenty-four hours after coronary ligation, Akap1-/- mice displayed larger infarct size compared to Siah2-/- or wt mice. One week after MI, cardiac function and survival were also significantly reduced in Akap1-/- mice, while cardiac fibrosis was significantly increased. Akap1 deletion was associated with remarkable mitochondrial structural abnormalities at electron microscopy, increased ROS production and reduced mitochondrial function after MI. These alterations were associated with enhanced cardiac mitophagy and apoptosis. Autophagy inhibition by 3-methyladenine significantly reduced apoptosis and ameliorated cardiac dysfunction following MI in Akap1-/- mice. These results demonstrate that Akap1 deficiency promotes cardiac mitochondrial aberrations and mitophagy, enhancing infarct size, pathological cardiac remodeling and mortality under ischemic conditions. Thus, mitochondrial AKAPs might represent important players in the development of post-ischemic cardiac remodeling and novel therapeutic targets.

3,5-Diiodo-L-thyronine activates brown adipose tissue thermogenesis in hypothyroid rats.

3,5-Diiodo-l-thyronine (T2), a thyroid hormone derivative, is capable of increasing energy expenditure, as well as preventing high fat diet-induced overweight and related metabolic dysfunction. Most studies to date on T2 have been carried out on liver and skeletal muscle. Considering the role of brown adipose tissue (BAT) in energy and metabolic homeostasis, we explored whether T2 could activate BAT thermogenesis. Using euthyroid, hypothyroid, and T2-treated hypothyroid rats (all maintained at thermoneutrality) in morphological and functional studies, we found that hypothyroidism suppresses the maximal oxidative capacity of BAT and thermogenesis, as revealed by reduced mitochondrial content and respiration, enlarged cells and lipid droplets, and increased number of unilocular cells within the tissue. In vivo administration of T2 to hypothyroid rats activated BAT thermogenesis and increased the sympathetic innervation and vascularization of tissue. Likewise, T2 increased BAT oxidative capacity in vitro when added to BAT homogenates from hypothyroid rats. In vivo administration of T2 to hypothyroid rats enhanced mitochondrial respiration. Moreover, UCP1 seems to be a molecular determinant underlying the effect of T2 on mitochondrial thermogenesis. In fact, inhibition of mitochondrial respiration by GDP and its reactivation by fatty acids were greater in mitochondria from T2-treated hypothyroid rats than untreated hypothyroid rats. In vivo administration of T2 led to an increase in PGC-1α protein levels in nuclei (transient) and mitochondria (longer lasting), suggesting a coordinate effect of T2 in these organelles that ultimately promotes net activation of mitochondrial biogenesis and BAT thermogenesis. The effect of T2 on PGC-1α is similar to that elicited by triiodothyronine. As a whole, the data reported here indicate T2 is a thyroid hormone derivative able to activate BAT thermogenesis.

Peptide gH625 enters into neuron and astrocyte cell lines and crosses the blood-brain barrier in rats.

Peptide gH625, derived from glycoprotein H of herpes simplex virus type 1, can enter cells efficiently and deliver a cargo. Nanoparticles armed with gH625 are able to cross an in vitro model of the blood-brain barrier (BBB). In the present study, in vitro experiments were performed to investigate whether gH625 can enter and accumulate in neuron and astrocyte cell lines. The ability of gH625 to cross the BBB in vivo was also evaluated. gH625 was administered in vivo to rats and its presence in the liver and in the brain was detected. Within 3.5 hours of intravenous administration, gH625 can be found beyond the BBB in proximity to cell neurites. gH625 has no toxic effects in vivo, since it does not affect the maximal oxidative capacity of the brain or the mitochondrial respiration rate. Our data suggest that gH625, with its ability to cross the BBB, represents a novel nanocarrier system for drug delivery to the central nervous system. These results open up new possibilities for direct delivery of drugs into patients in the field of theranostics and might address the treatment of several human diseases.

TRC150094 attenuates progression of nontraditional cardiovascular risk factors associated with obesity and type 2 diabetes in obese ZSF1 rats.

Chronic overnutrition and consequential visceral obesity is associated with a cluster of risk factors for cardiovascular disease and type 2 diabetes mellitus. Moreover, individuals who have a triad of hypertension, dysglycemia, and elevated triglycerides along with reduced high-density lipoprotein cholesterol have a greater residual cardiovascular risk even after factoring for the traditional risk factors such as age, smoking, diabetes, and elevated low-density lipoprotein cholesterol. In our previous study we demonstrated that TRC150094, when administered to rats receiving a high-fat diet, stimulated mitochondrial fatty acid oxidation (FAO) and reduced visceral adiposity, opening an interesting perspective for a possible clinical application. In the present study, oral administration of TRC150094 to obese Zucker spontaneously hypertensive fatty rats (obese ZSF1) improved glucose tolerance and glycemic profile as well as attenuated a rise in blood pressure. Obese ZSF1 rats treated with TRC150094 also showed reduced hepatic steatosis, reduced progression of nephropathy, and improved skeletal muscle function. At the cellular level, TRC150094 induced a significant increase in mitochondrial respiration as well as an increased FAO in liver and skeletal muscle, ultimately resulting in reduced hepatic as well as total body fat accumulation, as evaluated by magnetic resonance spectroscopy and magnetic resonance imaging, respectively. If reproduced in humans, these results could confirm that TRC150094 may represent an attractive therapeutic agent to counteract multiple residual cardiovascular risk components.