Oxidative damage and mitochondrial functionality in hearts from KO UCP3 mice housed at thermoneutrality

The antioxidant role of mitochondrial uncoupling protein 3 (UCP3) is controversial. This work aimed to investigate the effects of UCP3 on the heart of mice housed at thermoneutral temperature, an experimental condition that avoids the effects of thermoregulation on mitochondrial activity and redox homeostasis, preventing the alterations related to these processes from confusing the results caused by the lack of UCP3. WT and KO UCP3 mice were acclimatized at 30 °C for 4 weeks and hearts were used to evaluate metabolic capacity and redox state. Tissue and mitochondrial respiration, the activities of the mitochondrial complexes, and the protein expression of mitochondrial complexes markers furnished information on mitochondrial functionality. The levels of lipid and protein oxidative damage markers, the activity of antioxidant enzymes, the reactive oxygen species levels, and the susceptibility to in vitro Fe-ascorbate-induced oxidative stress furnished information on redox state. UCP3 ablation reduced tissue and mitochondrial respiratory capacities, not affecting the mitochondrial content. In KO UCP3 mice, the mitochondrial complexes activities were lower than in WT without changes in their content. These effects were accompanied by an increase in the level of oxidative stress markers, ROS content, and in vitro susceptibility to oxidative stress, notwithstanding that the activities of antioxidant enzymes were not affected by UCP3 ablation. Such modifications are also associated with enhanced activation/phosphorylation of EIF2α, a marker of integrated stress response and endoplasmic reticulum stress (GRP778 BIP). The lack of UCP3 makes the heart more prone to oxidative insult by reducing oxygen consumption and increasing ROS. Our results demonstrate that UCP3 helps the cell to preserve mitochondrial function by mitigating oxidative stress

Hepatitis C Virus Infection: Evidence for an Association With Type 2 Diabetes: Response to Skowroński et al.

We agree with Skowronski et al. (1) that the type of diabetes manifested by patients with HCV chronic infection (HCV+) may not be classical type 2 diabetes, and the phenotypic characterization of our patients shows just that. The labeling of HCV+ patients as type 2 diabetes is purely conventional and possibly inaccurate: the lines separating type 1 diabetes, from latent autoimmune diabetes in

Peroxisome Proliferator-Activated Receptor Delta: A Conserved Director of Lipid Homeostasis through Regulation of the Oxidative Capacity of Muscle

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

Hepatitis C Virus Infection: Evidence for an Association With Type 2 Diabetes

An increased prevalence of type 2 diabetes and impaired glucose tolerance has been consistently found in liver cirrhosis from any cause (1–3). Less clear is whether hepatitis C virus (HCV) infection is associated with type 2 diabetes in the absence of cirrhosis. Several reports have claimed a specific association between HCV infection and type 2 diabetes, but in most instances, patients were a mixture of cases with cirrhosis and hepatitis (4–6). Two clinic-based studies found an excess of type 2 diabetes in noncirrhotic HCV+ (NC-HCV+) patients compared with patients with chronic hepatitis of other origin (7–9), but another large study could not detect it (10). Furthermore, one clinic-based small study found a specific association with type 2 diabetes in NC-HCV+ patients (11) compared with a general population sample. The aim of this study was to establish the prevalence and clinical phenotype of type 2 diabetes in a large series of NC-HCV+ patients. A sample of the general population or patients with hepatitis B virus (HBV)-related noncirrhotic chronic hepatitis (NC-HBV+) was used as control subjects. From January 1995 to December 2001, 564 NC-HCV+ patients were consecutively examined at our center (none had been previously treated with interferon). Diagnosis of HCV infection was based on abnormal serum aminotransferases levels of >6 months' duration and positive testing for serum anti-HCV markers and

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

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

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

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

3,5-Diiodo-L-Thyronine Modifies the Lipid Droplet Composition in a Model of Hepatosteatosis

Background/Aims: Fatty acids are the main energy stores and the major membrane components of the cells. In the hepatocyte, fatty acids are esterified to triacylglycerols (TAGs) and stored in lipid droplets (LDs). The lipid lowering action of 3,5-diiodo-L-thyronine (T 2 ) on an in vitro model of hepatosteatosis was investigated in terms of fatty acid and protein content of LDs, lipid oxidation and secretion. Methods: FaO cells were exposed to oleate/ palmitate, then treated with T 2 . Results: T 2 reduced number and size of LDs, and modified their acyl composition by decreasing the content of saturated (SFA) vs monounsaturated (MUFA) fatty acids thus reversing the SFA/MUFA ratio. The expression of the LD-associated proteins adipose differentiation-related protein (ADRP), oxidative tissue-enriched PAT protein (OXPAT), and adipose triglyceride lipase (ATGL) was increased in 'steatotic' cells and further up-regulated by T 2 . Moreover, T 2 stimulated the mitochondrial oxidation by up-regulating carnitine-palmitoyl-transferase (CPT1), uncoupling protein 2 (UCP2) and very long-chain acylcoenzyme A dehydrogenase (VLCAD). Conclusions: T 2 leads to mobilization of TAGs from LDs and stimulates mitochondrial oxidative metabolism of fatty acids, in particular of SFAs, and thus enriches of MUFAs the LDs. This action may protect the hepatocyte from excess of SFAs that are more toxic than MUFAs

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

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

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