Mitochondrial Uncoupling Protein Research Articles

Acupuncture modulates the AMPK/PGC-1 signaling pathway to facilitate mitochondrial biogenesis and neural recovery in ischemic stroke rats.

The main objective of this study was to investigate the role and mechanism of acupuncture on anti-nerve injury in the acute phase by regulating mitochondrial energy metabolism via monophosphate-activated protein kinase (AMPK)/peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) axis in rat ischemic stroke. Middle cerebral artery occlusion (MCAO) was established by middle cerebral artery occlusion/reperfusion. One-week of acupuncture was performed during the acute phase of ischemic stroke. The neurological function and brain tissue integrity were evaluated. Mitochondrial function (intracellular ATP level and the activity of mitochondrial respiratory chain complex I) and the level of NADH oxidase (NOX) were detected by enzymatic chemistry. Next, the potential molecular mechanisms were explored by western blotting, fluorescence quantitative PCR and immunohistochemistry method. (1) Acupuncture treatment for MCAO/R rats showed a significant improvement in the infarcted tissue accompanied by functional recovery in Zea-Longa score and balance beam score outcomes, motor function performances. (2) Acupuncture increased the levels of ATP and mitochondrial respiratory chain complex I, decreased the NOX levels in cerebral ischemia established by suture-occluded method. (3) Acupuncture reduced the necrosis dissolution of neuronal cells and meningeal edema, while promoting angiogenesis. (4) Quantitative immunohistochemical staining results showed acupuncture can increase the expression of AMPK, p-AMPK and the mitochondrial transcription factor PGC-1α, NRF2, TFAM and uncoupling protein 2 (UCP2). Meanwhile, acupuncture treatment up-regulated the expression of the corresponding protein. (5) Subsequently, acupuncture enhanced AMPK phosphorylation as well as the expression of PGC-1α, NRF2, TFAM and UCP2, implicated in mitochondrial synthesis and cellular apoptosis. (6) Finally, injections of AMPK antagonists and activators confirmed AMPK as a therapeutic target for the anti-nerve damage effects of acupuncture. Acupuncture intervention relieved ischemic stroke progression in MCAO rats by promoting energy metabolism and mitochondrial biogenesis in the brain and alleviating neuronal apoptosis, which was mediated by eliciting AMPK/PGC-1α axis, among them AMPK is a therapeutic target.

Angiotensin II participates in mitochondrial thermogenic functions via the activation of glycolysis in chemically induced human brown adipocytes

Brown adipocytes are potential therapeutic targets for the prevention of obesity-associated metabolic diseases because they consume circulating glucose and fatty acids for heat production. Angiotensin II (Ang II) peptide is involved in the pathogenesis of obesity- and cold-induced hypertension; however, the mechanism underlying the direct effects of Ang II on human brown adipocytes remains unclear. Our transcriptome analysis of chemical compound-induced brown adipocytes (ciBAs) showed that the Ang II type 1 receptor (AGTR1), but not AGTR2 and MAS1 receptors, was expressed. The Ang II/AGTR1 axis downregulated the expression of mitochondrial uncoupling protein 1 (UCP1). The simultaneous treatment with β-adrenergic receptor agonists and Ang II attenuated UCP1 expression, triglyceride lipolysis, and cAMP levels, although cAMP response element-binding protein (CREB) phosphorylation was enhanced by Ang II mainly through the protein kinase C pathway. Despite reduced lipolysis, both coupled and uncoupled mitochondrial respiration was enhanced in Ang II-treated ciBAs. Instead, glycolysis and glucose uptake were robustly activated upon treatment with Ang II without a comprehensive transcriptional change in glucose metabolic genes. Elevated mitochondrial energy status induced by Ang II was likely associated with UCP1 repression. Our findings suggest that the Ang II/AGTR1 axis participates in mitochondrial thermogenic functions via glycolysis.

Ameliorative effect of bofutsushosan (Fangfengtongshengsan) extract on the progression of aging-induced obesity.

This study aimed to compare fat accumulation in young and aged mice raised on a high-fat diet and to characterize the obesity-reducing effects of a Kampo medicine, bofutsushosan (BTS; fangfengtongshengsan in Chinese). Aged mice fed a high-fat diet containing 2% BTS extract for 28days exhibited a significant reduction in weight gain and accumulation of visceral and subcutaneous fat, which were greater degree of reduction than those of the young mice. When the treatment period was extended to two months, the serum aspartate aminotransferase and alanine aminotransferase levels and the accumulation of fat droplets in the hepatocytes decreased. The mRNA expression of mitochondrial uncoupling protein 1 (UCP1) in the brown adipose tissue was significantly reduced in the aged mice compared to the young mice but increased by 2% in the BTS-treated aged mice. Additionally, the effect of BTS extract on oleic acid-albumin-induced triglyceride accumulation in hepatoblastoma-derived HepG2 cells was significantly inhibited in a concentration-dependent manner. Evaluation of the single crude drug extracts revealed that Forsythia Fruit, Schizonepeta Spike, and Rhubarb were the active components in BTS extract. These results suggest that BTS extract is effective against visceral, subcutaneous, and ectopic fats in the liver, which tend to accumulate with aging. Thus, BTS extract is useful in preventing and ameliorating the development of obesity and metabolic syndrome.

Mitochondrial uncoupling proteins protect human airway epithelial ciliated cells from oxidative damage

Apical cilia on epithelial cells defend the lung by propelling pathogens and particulates out of the respiratory airways. Ciliated cells produce ATP that powers cilia beating by densely grouping mitochondria just beneath the apical membrane. However, this efficient localization comes at a cost because electrons leaked during oxidative phosphorylation react with molecular oxygen to form superoxide, and thus, the cluster of mitochondria creates a hotspot for oxidant production. The relatively high oxygen concentration overlying airway epithelia further intensifies the risk of generating superoxide. Thus, airway ciliated cells face a unique challenge of producing harmful levels of oxidants. However, surprisingly, highly ciliated epithelia produce less reactive oxygen species (ROS) than epithelia with few ciliated cells. Compared to other airway cell types, ciliated cells express high levels of mitochondrial uncoupling proteins, UCP2 and UCP5. These proteins decrease mitochondrial protonmotive force and thereby reduce production of ROS. As a result, lipid peroxidation, a marker of oxidant injury, decreases. However, mitochondrial uncoupling proteins exact a price for decreasing oxidant production; they decrease the fraction of mitochondrial respiration that generates ATP. These findings indicate that ciliated cells sacrifice mitochondrial efficiency in exchange for safety from damaging oxidation. Employing uncoupling proteins to prevent oxidant production, instead of relying solely on antioxidants to decrease postproduction oxidant levels, may offer an advantage for targeting a local area of intense ROS generation.

Differential responses to UCP1 ablation in classical brown versus beige fat, despite a parallel increase in sympathetic innervation

In the cold, the absence of the mitochondrial uncoupling protein 1 (UCP1) results in hyper-recruitment of beige fat, but classical brown fat becomes atrophied. Here we examine possible mechanisms underlying this phenomenon. We confirm that in brown fat from UCP1-knockout (UCP1-KO) mice acclimated to the cold, the levels of mitochondrial respiratory chain proteins were diminished; however, in beige fat, the mitochondria seemed to be unaffected. The macrophages that accumulated massively not only in brown fat but also in beige fat of the UCP1-KO mice acclimated to cold did not express tyrosine hydroxylase, the norepinephrine transporter (NET) and monoamine oxidase-A (MAO-A). Consequently, they could not influence the tissues through the synthesis or degradation of norepinephrine. Unexpectedly, in the cold, both brown and beige adipocytes from UCP1-KO mice acquired an ability to express MAO-A. Adipose tissue norepinephrine was exclusively of sympathetic origin, and sympathetic innervation significantly increased in both tissues of UCP1-KO mice. Importantly, the magnitude of sympathetic innervation and the expression levels of genes induced by adrenergic stimulation were much higher in brown fat. Therefore, we conclude that no qualitative differences in innervation or macrophage character could explain the contrasting reactions of brown versus beige adipose tissues to UCP1-ablation. Instead, these contrasting responses may be explained by quantitative differences in sympathetic innervation: the beige adipose depot from the UCP1-KO mice responded to cold acclimation in a canonical manner and displayed enhanced recruitment, while the atrophy of brown fat lacking UCP1 may be seen as a consequence of supraphysiological adrenergic stimulation in this tissue.

Exercise-inducible circulating extracellular vesicle irisin promotes browning and the thermogenic program in white adipose tissue.

Exercise can reduce body weight and promote white fat browning, but the underlying mechanisms remain largely unknown. This study investigated the role of fibronectin type III domain-containing protein 5 (FNDC5)/Irisin, a hormone released from exercising muscle, in the browning of white fat in circulating extracellular vesicles (EVs). Mice were subjected to a 4 weeks of running table exercise, and fat browning was analyzed via histology, protein blotting and qPCR. Circulating EVs were extracted by ultrahigh-speed centrifugation, and ELISA was used to measure the irisin concentration in the circulating EVs. Circulating EVs that differentially expressed irisin were applied to adipocytes, and the effect of EV-irisin on adipocyte energy metabolism was analyzed by immunofluorescence, protein blotting, and cellular oxygen consumption rate analysis. During sustained exercise, the mice lost weight and developed fat browning. FNDC5 was induced, cleaved, and secreted into irisin, and irisin levels subsequently increased in the plasma during exercise. Interestingly, irisin was highly expressed in circulating EVs that effectively promoted adipose browning. Mechanistically, the circulating EV-irisin complex is transported intracellularly by the adipocyte membrane receptor integrin αV, which in turn activates the AMPK signaling pathway, which is dependent on mitochondrial uncoupling protein 1 to cause mitochondrial plasmonic leakage and promote heat production. After inhibition of the AMPK signaling pathway, the effects of the EV-irisin on promoting fat browning were minimal. Exercise leads to the accumulation of circulating EV-irisin, which enhances adipose energy metabolism and thermogenesis and promotes white fat browning in mice, leading to weight loss.

Modulation and Determination of the Status of Inflammasomes in Leishmania-Infected Macrophages.

Leishmania, an intra-macrophage kinetoplastid parasite, modulates a vast array of defensive mechanisms of the host macrophages to create a comfortable environment for their survival. When the host encounters intracellular pathogens, a multimeric protein complex called NLRP3 inflammasome gets turned on, leading to caspase-1 activation-mediated maturation of IL-1β from its pro-form. However, Leishmania often manages to neutralize inflammasome activation by manipulating negative regulatory molecules of the host itself. Exhaustion of NLRP3 and pro-IL-1β result from decreased NF-κB activity in infection, which was attributed to increased expression of A20, a negative regulator of NF-κB signalling. Moreover, reactive oxygen species, another key requirement for inflammasome activation, are inhibited by mitochondrial uncoupling protein 2 (UCP2) which is upregulated by Leishmania. Inflammasome activation is a complex event and procedures involved in monitoring inflammasome activation need to be accurate and error-free. In this chapter, we summarize the protocol that includes various experimental procedures required for the determination of the status of inflammasomes in Leishmania-infected macrophages.

MKP-2 Deficiency Leads to Lipolytic and Inflammatory Response to Fasting in Mice

MKP-2 Deficiency Leads to Lipolytic and Inflammatory Response to Fasting in Mice

High-throughput analysis reveals disturbances throughout the cell caused by Arabidopsis UCP1 and UCP3 double knockdown

Three genes encoding mitochondrial uncoupling proteins (UCPs) have been described in Arabidopsis thaliana (UCP1 to UCP3). In plants, UCPs may act as an uncoupler or as an aspartate/glutamate exchanger. For instance, much of the data regarding UCP functionality were obtained for the UCP1 and UCP2 isoforms compared with UCP3. Here, to get a better understanding about the concerted action of UCP1 and UCP3 in planta, we investigated the transcriptome and metabolome profiles of ucp1 ucp3 double mutant plants during the vegetative phase. For that, 21-day-old mutant plants, which displayed the most evident phenotypic alterations compared to wild type (WT) plants, were employed. The double knockdown of UCP1 and UCP3, isoforms unequivocally present inside the mitochondria, promoted important transcriptional reprogramming with alterations in the expression of genes related to mitochondrial and chloroplast function as well as those responsive to abiotic stress, suggesting disturbances throughout the cell. The observed transcriptional changes were well integrated with the metabolomic data of ucp1 ucp3 plants. Alterations in metabolites related to primary and secondary metabolism, particularly enriched in the Alanine, Aspartate and Glutamate metabolism, were detected. These findings extend our knowledge of the underlying roles played by UCP3 in concert with UCP1 at the whole plant level.

Human mitochondrial uncoupling protein 3 functions as a metabolite transporter.

Since its discovery, a major debate about mitochondrial uncoupling protein 3 (UCP3) has been whether its metabolic actions result primarily from mitochondrial inner membrane proton transport, a process that decreases respiratory efficiency and ATP synthesis. However, UCP3 expression and activity are induced by conditions that would seem at odds with inefficient 'uncoupled' respiration, including fasting and exercise. Here, we demonstrate that the bacterially expressed human UCP3, reconstituted into liposomes, catalyses a strict exchange of aspartate, malate, sulphate and phosphate. The R282Q mutation abolishes the transport activity of the protein. Although the substrate specificity and inhibitor sensitivity of UCP3 display similarity with that of its close homolog UCP2, the two proteins significantly differ in their transport mode and kinetic constants.

Mitochondrial uncoupling proteins regulate the metabolic function of human Sertoli cells.

Mitochondrial uncoupling proteins (UCPs) regulate mitochondrial activity and reactive oxygen species production through the transport of protons and metabolites. This study identified the expression of UCPs in human Sertoli cells, which proved to be modulators of their mitochondrial activity. Mitochondrial uncoupling proteins (UCPs) are mitochondrial channels responsible for the transport of protons and small molecular substrates across the inner mitochondrial membrane. Altered UCP expression or function is commonly associated with mitochondrial dysfunction and increased oxidative stress, which are both known causes of male infertility. However, UCP expression and function in the human testis remain to be characterized. This study aimed to assess the UCP homologs (UCP1-6) expression and function in primary cultures of human Sertoli cells (hSCs). We identified the mRNA expression of all UCP homologs (UCP1-6) and protein expression of UCP1, UCP2, and UCP3 in hSCs. UCP inhibition by genipin for 24 h decreased hSCs proliferation without causing cytotoxicity (n = 6). Surprisingly, the prolonged UCP inhibition for 24 h decreased mitochondrial membrane potential, oxygen consumption rate (OCR), and endogenous reactive oxygen species (ROS) production. The metabolism of hSCs was also affected as UCP inhibition shifted their metabolism toward an increased pyruvate consumption. Taken together, these findings demonstrate that UCPs play a role as regulators of the mitochondrial function in hSCs, emphasizing their potential as targets in the study of male (in)fertility.

Abstract 17359: UCP2 Induced Glycolysis Promotes Histone Acetylation to Regulate Cardiomyocyte Cell Cycle After Injury

Developmental cardiac tissue is proliferative capable of robust regenerative response after myocardial injury, but this ability is lost by P7 when cardiomyocytes (CMs) exit the cell cycle and become terminally differentiated. Postnatal CMs exhibit a specialized metabolic state that supports rapid proliferation and is altered with loss of regeneration in the heart. Studies have shown that reliance on glycolysis for energy generation is preferred by proliferating CMs and activation of glycolysis promotes cardiac repair. Nevertheless, the role of glycolysis in regulating CM gene program linked to cell cycle and repair in the heart remains poorly studied. Here, we identify a novel role for mitochondrial uncoupling protein 2 (UCP2) in the heart regulating CM cell cycle dynamics. UCP2 was found to be primarily active during cardiac development, rapidly decreases after birth and is expressed at low levels in the adult heart. UCP2 overexpression in human IPS derived CMs increases cell cycle progression as assessed by immunostaining and flow cytometry-based DNA content analysis. In parallel, neonatal rat ventricular myocytes (NRVMs) overexpressing UCP2 showed increased glycolysis and glycolytic metabolites, reduced mitochondrial membrane potential and mitochondrial activity compared to controls. Next, we generated αMHC-UCPKO mice that showed reduction in total number of CMs and pHH3 levels, increased CM size and ploidy during postnatal development. Adult αMHC-UCPKO subjected to myocardial infarction injury showed significant cardiac dysfunction and fibrosis at 4 weeks compared to controls. Mechanistically, UCP2 induced glycolysis increases ATP-citrate lyase (ACLY) mediated acetyl-CoA generation in the CMs. Additionally, CMs isolated from P1 αMHC-UCPKO showed several histone modifications including decrease in histone acetylation marks. Similarly, NRVMs overexpressing UCP2 showed increased histone H3 acetylation and expression of histone acetyltransferases (HATs) and corresponding decrease in histone deacetylases (HDACs). In conclusion, UCP2 is a novel developmental metabolic regulator of CM cell cycle in the neonatal and adult heart. Moreover, UCP2 induced glycolysis increases cell cycle via altering CM histone acetylation.

Specific Genotypes Associated with Differences in Fasting Insulin Levels and Body Mass Index in Healthy Young Males: Implications for Gene–Nutrient Interactions—an Exploratory Study

BackgroundGenetic variation may significantly impact an individual's susceptibility to diseases, particularly when combined with specific nutrients. Additionally, genetic variations can lead to interindividual differences in metabolic responses. ObjectiveThe present study explores the association between gene variants and observed interindividual differences in metabolic responses. MethodsThe study included 30 healthy males (aged 20–34) who underwent a fasting period and subsequently consumed a standardized meal. Blood samples were collected both before and after the meal to assess metabolic changes. BMI served as an indirect measure for assessing physiological responses associated with body composition. Appetite changes were assessed using an online Visual Analog 100-point Scale. Buccal swabs were collected to analyze genetic variants in single nucleotide polymorphisms (SNPs). ResultsThe data underwent multiple regression analysis, revealing significant associations with 3 SNPs and their metabolic status: the insulin-receptor substrate 1 (IRS1) gene variant rs2943641, genotypes CT and CC, with elevated fasting insulin levels (R2 = 0.639, P = < 0.0001); the mitochondrial uncoupling protein 1 (UCP1) gene variant rs1800592, genotypes GG and GA, with increased BMI (R2 = 0.261, P = 0.007); and the peroxisome proliferator-activated receptor γ2 (PPARγ2) gene variant rs1801282, genotypes GG and GC, with increased BMI (R2 = 0.200, P = 0.024). ConclusionsTherefore, our study established significant associations between these 3 SNPs and differences in fasting insulin levels and BMI within our cohort.

Plasmodium falciparum OPA3-like protein (PfOPA3) is essential for maintenance of mitochondrial homeostasis and parasite proliferation.

Metabolic pathways and proteins responsible for maintaining mitochondrial dynamics and homeostasis in the Plasmodium parasite, the causative agent of malaria, remain to be elucidated. Here, we identified and functionally characterized a novel OPA3-like domain-containing protein in P. falciparum (PfOPA3). We show that PfOPA3 is expressed in the intraerythrocytic stages of the parasite and localizes to the mitochondria. Inducible knock-down of PfOPA3 using GlmS ribozyme hindered the normal intraerythrocytic cycle of the parasites; specifically, PfOPA3-iKD disrupted parasite development as well as parasite division and segregation at schizont stages, which resulted in a drastic reduction in the number of merozoites progenies. Parasites lacking PfOPA3 show severe defects in the development of functional mitochondria; the mitochondria showed reduced activity of mtETC but not ATP synthesis, as evidenced by reduced activity of complex III of the mtETC, and increased sensitivity for drugs targeting DHODH as well as complex III, but not to the drugs targeting complex V. Further, PfOPA3 downregulation leads to reduction in the level of mitochondrial proton transport uncoupling protein (PfUCP) to compensate reduced activity of complex III and maintain proton efflux across the inner membrane. The reduced activity of DHODH, which is responsible for pyrimidine biosynthesis required for nuclear DNA synthesis, resulted in a significant reduction in parasite nuclear division and generation of progeny. In conclusion, we show that PfOPA3 is essential for the functioning of mtETC and homeostasis required for the development of functional mitochondria as well as for parasite segregation, and thus PfOPA3 is crucial for parasite survival during blood stages.

ACOT1 deficiency attenuates high-fat diet-induced fat mass gain by increasing energy expenditure.

Acyl-CoA thioesterase 1 (ACOT1) catalyzes the hydrolysis of long-chain acyl-CoAs to free fatty acids and CoA and is typically upregulated in obesity. Whether targeting ACOT1 in the setting of high-fat diet-induced (HFD-induced) obesity would be metabolically beneficial is not known. Here we report that male and female ACOT1KO mice are partially protected from HFD-induced obesity, an effect associated with increased energy expenditure without alterations in physical activity or food intake. In males, ACOT1 deficiency increased mitochondrial uncoupling protein-2 (UCP2) protein abundance while reducing 4-hydroxynonenal, a marker of oxidative stress, in white adipose tissue and liver of HFD-fed mice. Moreover, concurrent knockdown (KD) of UCP2 with ACOT1 in hepatocytes prevented increases in oxygen consumption observed with ACOT1 KD during high lipid loading, suggesting that UCP2-induced uncoupling may increase energy expenditure to attenuate weight gain. Together, these data indicate that targeting ACOT1 may be effective for obesity prevention during caloric excess by increasing energy expenditure.

Extracellular thiamine concentration influences thermogenic competency of differentiating neck area-derived human adipocytes.

Brown adipose tissue (BAT) dissipates energy in the form of heat majorly via the mitochondrial uncoupling protein 1 (UCP1). The activation of BAT, which is enriched in the neck area and contains brown and beige adipocytes in humans, was considered as a potential therapeutic target to treat obesity. Therefore, finding novel agents that can stimulate the differentiation and recruitment of brown or beige thermogenic adipocytes are important subjects for investigation. The current study investigated how the availability of extracellular thiamine (vitamin B1), an essential cofactor of mitochondrial enzyme complexes that catalyze key steps in the catabolism of nutrients, affects the expression of thermogenic marker genes and proteins and subsequent functional parameters during ex vivo adipocyte differentiation. We differentiated primary human adipogenic progenitors that were cultivated from subcutaneous (SC) or deep neck (DN) adipose tissues in the presence of gradually increasing thiamine concentrations during their 14-day differentiation program. mRNA and protein expression of thermogenic genes were analyzed by RT-qPCR and western blot, respectively. Cellular respiration including stimulated maximal and proton-leak respiration was measured by Seahorse analysis. Higher thiamine levels resulted in increased expression of thiamine transporter 1 and 2 both at mRNA and protein levels in human neck area-derived adipocytes. Gradually increasing concentrations of thiamine led to increased basal, cAMP-stimulated, and proton-leak respiration along with elevated mitochondrial biogenesis of the differentiated adipocytes. The extracellular thiamine availability during adipogenesis determined the expression levels of UCP1, PGC1a, CKMT2, and other browning-related genes and proteins in primary SC and DN-derived adipocytes in a concentration-dependent manner. Providing abundant amounts of thiamine further increased the thermogenic competency of the adipocytes. Case studies in humans reported that thiamine deficiency was found in patients with type 2 diabetes and obesity. Our study raises the possibility of a novel strategy with long-term thiamine supplementation, which can enhance the thermogenic competency of differentiating neck area-derived adipocytes for preventing or combating obesity.

Electroacupuncture intervention improves lipid metabolism and promotes browning of white adipose tissue by activating AMPK/Sirt1 pathway and up-regulating Nrg4 content in middle-aged and aged obese rats

To investigate the mechanism of electroacupuncture （EA） in promoting the browning of white adipose tissue in middle-aged and aged obese rats induced by high fat by regulating AMP-activated protein kinase （AMPK） /silence-information regulatory factor 1 （Sirt1） pathway and neuregulin 4 （Nrg4）. Twenty-four male SD rats were randomized into blank control, model and EA groups （n=8 per group）. The obesity model was established by feeding the rats with high-fat diet for 6 weeks. For the EA group, EA （2 Hz/15 Hz, 1.5 mA） was applied to "Guanyuan" （CV4） and bilateral "Shenshu" （BL23）, "Fenglong" （ST40） and "Tianshu" （ST25） for 20 min, once a day, 5 days a week for 6 weeks. Rats of the blank control and model groups were also restrained for 20 min. The body mass and food intake were measured every week, and the Lee's index, epididymal fat, perirenal fat and brown adipose tissue were weighed. The contents of serum total cholesterol （TC）, triglyceride （TG）, high density lipoprotein cholesterol （HDL-C）, low density lipoprotein cholesterol （LDL-C） and norepinephrine （NE） were determined by ELISA. H.E. staining was used to observe the morphological changes of white and brown adipose tissue. The mRNA expression levels of mitochondrial uncoupling protein 1 （UCP1）, peroxisome proliferator activated receptor γ co-activator 1α （PGC-1α）, PR-domain protein 16 （PRDM16）, peroxisome proliferator activated receptor γ （PPARγ） and Nrg4 in the adipose tissue were detected by quantitative real time PCR, and the protein expression levels of Nrg4, AMPKα, Sirt1 and interleukin-6 （IL-6） in the white and brown adipose tissue were detected by Western blot. Compared with the blank control group, the body mass, food intake, the Lee's index, epididymal fat and perirenal fat mass, and serum TG, TC and LDL-C contents and the expression level of IL-6 protein were significantly increased （P<0.01, P<0.05, P<0.001）, and the brown adipose mass, serum HDL-C and NE contents, the expression levels of UCP1, PGC-1α, PRDM16, PPARγ and Nrg4 mRNAs, and the protein expression levels of AMPKα, Sirt1 and Nrg4 proteins in both white and brown adipose tissues were significantly decreased in the model group （P<0.01, P<0.05）. After EA intervention, the increased levels of body mass, food intake, Lee's index, epididymal fat and perirenal fat mass, serum TG, TC and LDL-C contents, and the expression of IL-6 protein, and the decreased levels of brown adipose mass, serum HDL-C and NE contents, expression levels of UCP1, PGC-1α, PRDM16, PPARγ and Nrg4 mRNAs, and those of AMPKα, Sirt1 and Nrg4 proteins in both white and brown adipose tissues were apparently reversed（P<0.05, P<0.01, P<0.001）. H.E. staining showed an increase of the volume and content of intracellular vacuoles of both white and brown adipose tissues, disordered arrangement of cells with vague boundary in the model group, which was relatively milder including a decrease of volume and content of vacuoles of both white and brown adipose, neat arrangement of cells with clear boundary. EA intervention can improve lipid metabolism and promote white adipose tissue browning in middle-aged and aged obese rats, which is possibly associated with its functions in activating AMPK/Sirt1 signaling pathway and up-regulating the level of Nrg4.

Thermogenic Modulation of Adipose Depots: A Perspective on Possible Therapeutic Intervention with Early Cardiorenal Complications of Metabolic Impairment.

Cardiovascular complications of diabetes and obesity remain a major cause for morbidity and mortality worldwide. Despite significant advances in the pharmacotherapy of metabolic disease, the available approaches do not prevent or slow the progression of complications. Moreover, a majority of patients present with significant vascular involvement at early stages of dysfunction prior to overt metabolic changes. The lack of disease-modifying therapies affects millions of patients globally, causing a massive economic burden due to these complications. Significantly, adipose tissue inflammation was implicated in the pathogenesis of metabolic syndrome, diabetes, and obesity. Specifically, perivascular adipose tissue (PVAT) and perirenal adipose tissue (PRAT) depots influence cardiovascular and renal structure and function. Accumulating evidence implicates localized PVAT/PRAT inflammation as the earliest response to metabolic impairment leading to cardiorenal dysfunction. Increased mitochondrial uncoupling protein 1 (UCP1) expression and function lead to PVAT/PRAT hypoxia and inflammation as well as vascular, cardiac, and renal dysfunction. As UCP1 function remains an undruggable target so far, modulation of the augmented UCP1-mediated PVAT/PRAT thermogenesis constitutes a lucrative target for drug development to mitigate early cardiorenal involvement. This can be achieved either by subtle targeted reduction in UCP-1 expression using innovative proteolysis activating chimeric molecules (PROTACs) or by supplementation with cyclocreatine phosphate, which augments the mitochondrial futile creatine cycling and thus decreases UCP1 activity, enhances the efficiency of oxygen use, and reduces hypoxia. Once developed, these molecules will be first-in-class therapeutic tools to directly interfere with and reverse the earliest pathology underlying cardiac, vascular, and renal dysfunction accompanying the early metabolic deterioration. SIGNIFICANCE STATEMENT: Adipose tissue dysfunction plays a major role in the pathogenesis of metabolic diseases and their complications. Although mitochondrial alterations are common in metabolic impairment, it was only recently shown that the early stages of metabolic challenge involve inflammatory changes in select adipose depots associated with increased uncoupling protein 1 thermogenesis and hypoxia. Manipulating this mode of thermogenesis can help mitigate the early inflammation and the consequent cardiorenal complications.

Inhibition of pyroptosis and apoptosis by capsaicin protects against LPS-induced acute kidney injury through TRPV1/UCP2 axis in vitro.

Acute kidney injury is a fatal disease characterized by a rapid deterioration of kidney function. Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide) is a natural product extracted from Capsicum. The aim of this study was to explore the protective effect of capsaicin on inflammation, apoptosis, and mitochondrial dysfunction in an in vitro model of acute kidney injury. Lipopolysaccharide (LPS)-induced acute kidney injury model was established in HK-2 cells to investigate the protective effect of capsaicin. Cell viability was assessed using CCK-8 assay, and protein expression was detected using western blot and immunofluorescence assay. Intracellular reactive oxygen species (ROS) level and mitochondrial membrane potential were analyzed by flow cytometry. Cell apoptosis was detected by propidium iodide staining. The results showed that capsaicin ameliorated LPS-induced cytotoxicity in vitro and attenuated the release of interleukin (IL)-1β and IL-18. Intriguingly, genipin abolished the protective effect of capsaicin. Molecularly, capsaicin activated transient receptor potential cation channel subfamily V member 1 -mitochondrial uncoupling protein 2 axis and inhibited caspase-1-mediated pyroptosis. In addition, capsaicin alleviated LPS-induced ROS production and mitochondrial membrane potential disruption and inhibited apoptosis. These findings suggest that capsaicin shows a protective effect in in vitro acute kidney injury model.

Mammary duct luminal epithelium controls adipocyte thermogenic programme.

Sympathetic activation during cold exposure increases adipocyte thermogenesis via the expression of mitochondrial protein uncoupling protein 1(UCP1)1. The propensity of adipocytes to express UCP1 is under a critical influence of the adipose microenvironment and varies between sexes and among various fat depots2-7. Here we report that mammary gland ductal epithelial cells in the adipose niche regulate cold-induced adipocyte UCP1 expression in female mouse subcutaneous white adipose tissue (scWAT). Single-cell RNA sequencing shows that glandular luminal epithelium subtypes express transcripts that encode secretory factors controlling adipocyte UCP1 expression under cold conditions. We term these luminal epithelium secretory factors 'mammokines'. Using 3D visualization of whole-tissue immunofluorescence, we reveal sympathetic nerve-ductal contact points. We show that mammary ducts activated by sympathetic nerves limit adipocyte UCP1 expression via the mammokine lipocalin2. In vivo and ex vivo ablation of mammary duct epithelium enhance the cold-induced adipocyte thermogenic gene programme in scWAT. Since the mammary duct network extends throughout most of the scWAT in female mice, females show markedly less scWAT UCP1 expression, fat oxidation, energy expenditure and subcutaneous fat mass loss compared with male mice, implicating sex-specific roles of mammokines in adipose thermogenesis. These results reveal a role of sympathetic nerve-activated glandular epithelium in adipocyte UCP1 expression and suggest that mammary duct luminal epithelium has an important role in controlling glandular adiposity.