Trib1 deficiency causes brown adipose respiratory chain depletion and mitochondrial disorder

Tribbles homolog 1 (TRIB1) belongs to the Tribbles family of pseudokinases, which plays a key role in tumorigenesis and inflammation. Although genome-wide analysis shows that TRIB1 expression is highly correlated with blood lipid levels, the relationship between TRIB1 and adipose tissue metabolism remains unclear. Accordingly, the aim of the present study was to explore the role of TRIB1 on mitochondrial function in the brown adipose tissue (BAT). Trib1-knockout mice were established using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology. The metabolic function of the BAT was induced by a β3-adrenoceptor agonist and the energy metabolism function of mitochondria in the BAT of mice was evaluated. Trib1-knockout mice exhibited obesity and impaired BAT thermogenesis. In particular, Trib1 knockout reduced the ability of the BAT to maintain body temperature, inhibited β3-adrenoceptor agonist-induced thermogenesis, and accelerated lipid accumulation in the liver and adipose tissues. In addition, Trib1 knockout reduced mitochondrial respiratory chain complex III activity, produced an imbalance between mitochondrial fusion and fission, caused mitochondrial structural damage and dysfunction, and affected heat production and lipid metabolism in the BAT. Conversely, overexpression of Trib1 in 3T3-L1 adipocytes increased the number of mitochondria and improved respiratory function. These findings support the role of Trib1 in regulating the mitochondrial respiratory chain and mitochondrial dynamics by affecting mitochondrial function and thermogenesis in the BAT.Subject terms: Energy metabolism, Obesity     

Mfn2 is critical for brown adipose tissue thermogenic function

Mitochondrial fusion and fission events, collectively known as mitochondrial dynamics, act as quality control mechanisms to ensure mitochondrial function and fine‐tune cellular bioenergetics. Defective mitofusin 2 (Mfn2) expression and enhanced mitochondrial fission in skeletal muscle are hallmarks of insulin‐resistant states. Interestingly, Mfn2 is highly expressed in brown adipose tissue (BAT), yet its role remains unexplored. Using adipose‐specific Mfn2 knockout (Mfn2‐adKO) mice, we demonstrate that Mfn2, but not Mfn1, deficiency in BAT leads to a profound BAT dysfunction, associated with impaired respiratory capacity and a blunted response to adrenergic stimuli. Importantly, Mfn2 directly interacts with perilipin 1, facilitating the interaction between the mitochondria and the lipid droplet in response to adrenergic stimulation. Surprisingly, Mfn2‐adKO mice were protected from high‐fat diet‐induced insulin resistance and hepatic steatosis. Altogether, these results demonstrate that Mfn2 is a mediator of mitochondria to lipid droplet interactions, influencing lipolytic processes and whole‐body energy homeostasis.     

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.     

Brown adipose tissue mitochondria: modulation by GDP and fatty acids depends on the respiratory substrates

The UCP1 [first UCP (uncoupling protein)] that is found in the mitochondria of brown adipocytes [BAT (brown adipose tissue)] regulates the heat production, a process linked to non-shivering thermogenesis. The activity of UCP1 is modulated by GDP and fatty acids. In this report, we demonstrate that respiration and heat released by BAT mitochondria vary depending on the respiratory substrate utilized and the coupling state of the mitochondria. It has already been established that, in the presence of pyruvate/malate, BAT mitochondria are coupled by faf-BSA (fatty-acid-free BSA) and GDP, leading to an increase in ATP synthesis and mitochondrial membrane potential along with simultaneous decreases in both the rates of respiration and heat production. Oleate restores the uncoupled state, inhibiting ATP synthesis and increasing the rates of both respiration and heat production. We now show that in the presence of succinate: (i) the rates of uncoupled mitochondria respiration and heat production are five times slower than in the presence of pyruvate/malate; (ii) faf-BSA and GDP accelerate heat and respiration as a result and, in coupled mitochondria, these two rates are accelerated compared with pyruvate/malate; (iii) in spite of the differences in respiration and heat production noted with the two substrates, the membrane potential and the ATP synthesized were the same; and (iv) oleate promoted a decrease in heat production and respiration in coupled mitochondria, an effect different from that observed using pyruvate/malate. These effects are not related to the production of ROS (reactive oxygen species). We suggest that succinate could stimulate a new route to heat production in BAT mitochondria.     

Assessment of brown adipose tissue activity in rats by 99mTc-sestamibi uptake

Brown adipose tissue (BAT) physiology and imaging have recently attracted considerable attention. BAT is characterized both by enhanced perfusion and increased mitochondrial activity. (99m)Tc-sestamibi is a lipophilic cationic tracer that concentrates in mitochondria. Data on the accumulation of (99m)Tc-sestamibi in BAT are currently lacking. This study investigates the in vivo (99m)Tc-sestamibi uptake in rat BAT. (99m)Tc-sestamibi was administered in male Wistar rats of various age and body size. (99m)Tc-sestamibi uptake was measured in vitro in BAT and white fat (WF) together with cytochrome c oxidase activity. Both (99m)Tc-sestamibi uptake and cytochrome c oxidase activity were higher in BAT than in WF (P<0.05). (99m)Tc-Sestamibi uptake in both BAT and WF was negatively related to body weight (r = -0.96 and -0.89, respectively) as was the BAT/WF uptake ratio (r = -0.85). These data show a higher (99m)Tc-sestamibi uptake in BAT compared to WF, in agreement with the high mitochondrial content and respiratory activity of the former. The strong negative correlation between (99m)Tc-sestamibi uptake in BAT and body weight (negative allometry), is in accordance to increased needs of thermogenesis in smaller animals. Implications of increased (99m)Tc-sestamibi uptake in BAT in radionuclide imaging are also discussed.     

Evaluation of mitochondrial respiratory function in small biopsies of liver

Mitochondrial respiratory function was studied in permeabilized pig liver biopsies. The cell membrane was permeabilized mechanically in tissue samples of 2-7 mg, for application of a standardized substrate/inhibitor titration protocol in high-resolution respirometry. Specific respirometric tests demonstrated complete plasma membrane permeabilization and accessibility of substrates to intact mitochondria. High respiratory adenylate control ratios and cytochrome c conservation in the tissue preparation were comparable or even better than in isolated mitochondria. Citrate synthase and cytochrome c oxidase activities remained at 85% of controls after up to 98 h storage of liver tissue at 0 degrees C in histidine-tryptophan-ketoglutarate solution. Multiple mitochondrial defects, however, were indicated after 48 h cold storage by the decline in respiratory capacity, which was lowered to a larger extent with complex I substrates compared to respiration with substrates for complex II or IV, measured in the absence of cytochrome c. After prolonged ischemia, the adenylate control ratio was significantly reduced, and cytochrome c depletion was detected by the stimulatory effect of cytochrome c. High-resolution respirometry allows the assessment of mitochondrial function in a few milligrams of permeabilized liver tissue, without isolation of mitochondria. This provides a basis for the analysis of mitochondrial function in human liver biopsies.     

Gender-related differences in morphology and thermogenic capacity of brown adipose tissue mitochondrial subpopulations

To investigate the possible existence of a gender dimorphism in the morphology and functionality of brown adipose tissue (BAT) mitochondrial subpopulations, we obtained three mitochondrial fractions - heavy, medium and light - by differential centrifugation. Electron microscopic analysis was carried out and mitochondrial protein content, cytochrome c oxidase and ATP synthase activities, mitochondrial DNA content and UCP1 protein levels were measured in each mitochondrial fraction. Female rats showed a greater mitochondrial size than males, with a different distribution pattern of the subpopulations. These differences were accompanied by higher oxidative and thermogenic capacities and a higher protein content in female rat BAT. This tissue also showed a greater tendency to respiratory chain uncoupling, as well as a close coordination between the oxidative, phosphorylative and thermogenic processes. These differences were found in the heavy subpopulation but not in the light one. Our results demonstrate that female rat BAT shows a highly differentiated mitochondrial pool, with the heavy mitochondrial subpopulation as the main responsible for the greater thermogenic activity of this tissue. In addition, it seems that there is a differential regulation of the mitochondrial growth cycle between genders in BAT, which leads to enhanced thermogenic capacity in female rat mitochondria.     

Tempol protection of spinal cord mitochondria from peroxynitrite-induced oxidative damage

Peroxynitrite (PN)-mediated mitochondrial dysfunction has been implicated in the secondary injury process after traumatic spinal cord injury (SCI). This study investigated the detrimental effects of the PN donor SIN-1 (3-morpholinosydnonimine) on isolated healthy spinal cord mitochondria and the protective effects of tempol, a catalytic scavenger of PN-derived radicals. A 5 min exposure of the mitochondria to SIN-1 caused a dose-dependent decrease in the respiratory control ratio (RCR) that was accompanied by significant increases in complex I-driven states II and IV respiration rates and decreases in states III and V. These impairments occurred together with an increase in mitochondrial protein 3-nitrotyrosine (3-NT), but not in lipid peroxidation (LP)-related 4-hydroxynonenal (4-HNE). Tempol significantly antagonized the respiratory effects of SIN-1 in parallel with an attenuation of 3-NT levels. These results show that the exogenous PN donor, SIN-1, rapidly causes mitochondrial oxidative damage and complex I dysfunction identical to traumatic spinal cord mitochondrial impairment and that this is mainly due to tyrosine nitration. Consistent with that, the protection of mitochondrial respiratory function by tempol is associated with a decrease in 3-NT levels in mitochondrial proteins also similar to the previously reported antioxidant actions of tempol in traumatically-injured spinal cord mitochondria.     

Cold-induced activation of brown adipose tissue and adipose angiogenesis in mice

Exposure of humans and rodents to cold activates thermogenic activity in brown adipose tissue (BAT). This protocol describes a mouse model to study the activation of BAT and angiogenesis in adipose tissues by cold acclimation. After a 1-week exposure to 4 °C, adult C57BL/6 mice show an obvious transition from subcutaneous white adipose tissue (WAT) into brown-like adipose tissue (BRITE). The BRITE phenotype persists after continuous cold exposure, and by the end of week 5 BRITE contains a high number of uncoupling protein-1-positive mitochondria, a characteristic feature of BAT. During the transition from WAT into BRITE, the vascular density is markedly increased owing to the activation of angiogenesis. In BAT, cold exposure stimulates thermogenesis by increasing the mitochondrial content and metabolic rate. BAT and the increased metabolic rate result in a lean phenotype. This protocol provides an outstanding opportunity to study the molecular mechanisms that control adipose mass.     

Evaluation of basic mitochondrial functions using rat tissue homogenates

The primary attempt in diagnostic and experimental studies of numerous pathological states associated with mitochondrial dysfunction is a precise evaluation of changes in function, content and structure of mitochondrial OXPHOS system. The analysis of rat heart, liver, brain and kidney by oxygraphy, enzyme activities, membrane potential, and BN/SDS-PAGE western blotting demonstrated that tissue homogenates can substitute for isolated mitochondria, providing comparable qualitative mitochondrial parameters. The use of homogenate avoids the loss of the majority of mitochondria during their isolation. Only 50–100 mg of the tissue is required for the complex OXPHOS analysis, i.e. five times less as compared with isolated mitochondria.     

Sources of heat during nonshivering thermogenesis in Djungarian hamsters: a dominant role of brown adipose tissue during cold adaptation

Summary To assess the thermogenic importance of BAT in Djungarian hamsters we removed about 40% of their BAT and compared their thermogenic abilities before and after the operation. BAT was weighed and assayed for its respiratory properties (Cox, mitochondria). Following removal of BAT we observed considerable reductions of NST. The comparison of NST with BAT weight and with respiratory properties of BAT following partial removal of BAT revealed that at least three different pathways for heat production were involved in NST. In cold-adapted hamsters (values for warm-adapted hamsters in parentheses) we estimated that 66.2% (37.0%) of all NST was produced by mitochondrial respiration in BAT; 16.3% (38.4%) was produced in other organ sites but required the presence of BAT, i.e. there was a mediatory action of BAT on thermogenesis in other organ sites. A further 11.5% (23%) of NST occurred outside of and independent of BAT. Mitochondrial respiration in BAT was the only compartment of NST which increased its contribution during cold adaptation (238 mW to 1,062 mW), whereas the other sources of heat remained largely unchanged.Abbreviations BAT brown adipose tissue - BATex partial removal of brown adipose tissue - BMR basal metabolic rate at thermoneutrality - Cox cytochrome c oxidase - NA noradrenaline - NST nonshivering thermogenesis     

Antioxidant properties of UCP1 are evolutionarily conserved in mammals and buffer mitochondrial reactive oxygen species

Mitochondrial uncoupling reduces reactive oxygen species (ROS) production and appears to be important for cellular signaling/protection, making it a focus for the treatment of metabolic and age-related diseases. Whereas the physiological role of uncoupling protein 1 (UCP1) of brown adipose tissue is established for thermogenesis, the function of UCP1 in the reduction of ROS in cold-exposed animals is currently under debate. Here, we investigated the role of UCP1 in mitochondrial ROS handling in the Lesser hedgehog tenrec (Echinops telfairi), a unique protoendothermic Malagasy mammal with recently identified brown adipose tissue (BAT). We show that the reduction of ROS by UCP1 activity also occurs in BAT mitochondria of the tenrec, suggesting that the antioxidative role of UCP1 is an ancient mammalian trait. Our analysis shows that the quantity of UCP1 displays strong control over mitochondrial hydrogen peroxide release, whereas other factors, such as mild cold, nonshivering thermogenesis, oxidative capacity, and mitochondrial respiration, do not correlate. Furthermore, hydrogen peroxide release from recoupled BAT mitochondria was positively associated with mitochondrial membrane potential. These findings led to a model of UCP1 controlling mitochondrial ROS release and, presumably, being controlled by high membrane potential, as proposed in the canonical model of “mild uncoupling”. Our study further promotes a conserved role for UCP1 in the prevention of oxidative stress, which was presumably established during evolution before UCP1 was physiologically integrated into nonshivering thermogenesis.     

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose tissue (WAT) by its discrete location and a brown-red color due to rich vascularization and high density of mitochondria. BAT plays a major role in energy expenditure and non-shivering thermogenesis in newborn mammals as well as the adults ¹. BAT-mediated thermogenesis is highly regulated by the sympathetic nervous system, predominantly via β adrenergic receptor ^{2, 3}. Recent studies have shown that BAT activities in human adults are negatively correlated with body mass index (BMI) and other diabetic parameters ^4-6. BAT has thus been proposed as a potential target for anti-obesity/anti-diabetes therapy focusing on modulation of energy balance ^6-8. While several cold challenge-based positron emission tomography (PET) methods are established for detecting human BAT ^9-13, there is essentially no standardized protocol for imaging and quantification of BAT in small animal models such as mice. Here we describe a robust PET/CT imaging method for functional assessment of BAT in mice. Briefly, adult C57BL/6J mice were cold treated under fasting conditions for a duration of 4 hours before they received one dose of ¹⁸F-Fluorodeoxyglucose (FDG). The mice were remained in the cold for one additional hour post FDG injection, and then scanned with a small animal-dedicated micro-PET/CT system. The acquired PET images were co-registered with the CT images for anatomical references and analyzed for FDG uptake in the interscapular BAT area to present BAT activity. This standardized cold-treatment and imaging protocol has been validated through testing BAT activities during pharmacological interventions, for example, the suppressed BAT activation by the treatment of β-adrenoceptor antagonist propranolol ^{14, 15}, or the enhanced BAT activation by β3 agonist BRL37344 ¹⁶. The method described here can be applied to screen for drugs/compounds that modulate BAT activity, or to identify genes/pathways that are involved in BAT development and regulation in various preclinical and basic studies.     

Simultaneous evaluation of substrate-dependent oxygen consumption rates and mitochondrial membrane potential by TMRM and safranin in cortical mitochondria

Mitochondrial membrane potential (mtMP) is critical for maintaining the physiological function of the respiratory chain to generate ATP. The present study characterized the inter-relationship between mtMP, using safranin and tetramethyl rhodamine methyl ester (TMRM), and mitochondrial respiratory activity and established a protocol for functional analysis of mitochondrial bioenergetics in a multi-sensor system. Coupled respiration was decreased by 27 and 30–35% in the presence of TMRM and safranin respectively. Maximal respiration was higher than coupled with Complex I- and II-linked substrates in the presence of both dyes. Safranin showed decreased maximal respiration at a higher concentration of carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) compared with TMRM. FCCP titration revealed that maximal respiration in the presence of glutamate and malate was not sustainable at higher FCCP concentrations as compared with pyruvate and malate. Oxygen consumption rate (OCR) and mtMP in response to mitochondrial substrates were higher in isolated mitochondria compared with tissue homogenates. Safranin exhibited higher sensitivity to changes in mtMP than TMRM. This multi-sensor system measured mitochondrial parameters in the brain of transgenic mice that model Alzheimer''s disease (AD), because mitochondrial dysfunction is believed to be a primary event in the pathogenesis of AD. The coupled and maximal respiration of electron transport chain were decreased in the cortex of AD mice along with the mtMP compared with age-matched controls. Overall, these data demonstrate that safranin and TMRM are suitable for the simultaneous evaluation of mtMP and respiratory chain activity using isolated mitochondria and tissue homogenate. However, certain care should be taken concerning the selection of appropriate substrates and dyes for specific experimental circumstances.     

Interferon αβ-induced abnormalities in adipocytes of suckling mice

The modification induced by interferon (IFN) in brown adipose tissue (BAT) was studied by high spatial resolution magnetic resonance imaging (MRI), histology, and transmission electron microscopy (TEM). In IFN-treated mice, at MRI, the interscapular BAT was slightly enlarged and showed non-homogeneous areas of lipid accumulation. The thickness of the subcutaneous white adipose tissue was reduced with respect to control mice. In the liver, MRI showed a lipid accumulation. In IFN-treated mice, by light microscopy, brown adipocytes showed a larger lipid deposit with respect to control mice. At TEM, in BAT, the mitochondria were reduced in number, smaller and the number of cristae was also significantly reduced with respect to the controls (9.1 ± 1.5 vs 20.1 ± 1.9, P < 0.01). The inclusions in the mitochondrial matrix were significantly less numerous in IFN-treated than in control animals (1.9 ± 0.7 vs 0.9 ± 0.7 for mitochondrial section, P < 0.01). Abnormalities of endoplasmic reticulum described in hepatocytes were not found in brown adipocytes of IFN-treated mice. The present work demonstrates that, in the BAT of sucking mice, IFN-treatment induces morphologic alterations and that brown adipocytes have MRI and TEM features resembling those found in the lipid laden BAT of aged animals.     

Uncoupling Protein 1 Decreases Superoxide Production in Brown Adipose Tissue Mitochondria

In thermogenic brown adipose tissue, uncoupling protein 1 (UCP1) catalyzes the dissipation of mitochondrial proton motive force as heat. In a cellular environment of high oxidative capacity such as brown adipose tissue (BAT), mitochondrial uncoupling could also reduce deleterious reactive oxygen species, but the specific involvement of UCP1 in this process is disputed. By comparing brown adipose tissue mitochondria of wild type mice and UCP1-ablated litter mates, we show that UCP1 potently reduces mitochondrial superoxide production after cold acclimation and during fatty acid oxidation. We address the sites of superoxide production and suggest diminished probability of “reverse electron transport” facilitated by uncoupled respiration as the underlying mechanism of reactive oxygen species suppression in BAT. Furthermore, ablation of UCP1 represses the cold-stimulated increase of substrate oxidation normally seen in active BAT, resulting in lower superoxide production, presumably avoiding deleterious oxidative damage. We conclude that UCP1 allows high oxidative capacity without promoting oxidative damage by simultaneously lowering superoxide production.     

In vitro brown and “brite”/“beige” adipogenesis: Human cellular models and molecular aspects

Brown adipose tissue (BAT) has long been thought to be absent or very scarce in human adults so that its contribution to energy expenditure was not considered as relevant. The recent discovery of thermogenic BAT in human adults opened the field for innovative strategies to combat overweight/obesity and associated diseases. This energy-dissipating function of BAT is responsible for adaptive thermogenesis in response to cold stimulation. In this context, adipocytes can be converted, within white adipose tissue (WAT), into multilocular adipocytes expressing UCP1, a mitochondrial protein that plays a key role in heat production by uncoupling the activity of the respiratory chain from ATP synthesis. These adipocytes have been named “brite” or “beige” adipocytes. Whereas BAT has been studied for a long time in murine models both in vivo and in vitro, there is now a strong demand for human cellular models to validate and/or identify critical factors involved in the induction of a thermogenic program within adipocytes. In this review we will discuss the different human cellular models described in the literature and what is known regarding the regulation of their differentiation and/or activation process. In addition, the role of microRNAs as novel regulators of brown/“brite” adipocyte differentiation and conversion will be depicted. Finally, investigation of both the conversion and the metabolism of white-to-brown converted adipocytes is required for the development of therapeutic strategies targeting overweight/obesity and associated diseases. This article is part of a Special Issue entitled Brown and White Fat: From Signaling to Disease.