Uncoupling Protein Research Articles

Oxaloacetic acid induces muscle energy substrate depletion and fatigue by JNK-mediated mitochondrial uncoupling.

Fatigue is a common phenomenon closely related to physical discomfort and numerous diseases, which is severely threatening the life quality and health of people. However, the exact mechanisms underlying fatigue are not fully characterized. Herein, we demonstrate that oxaloacetic acid (OAA), a crucial tricarboxylic acid cycle intermediate, modulates the muscle fatigue. The results showed that serum OAA level was positively correlated with fatigue state of mice. OAA-treated induced muscle fatigue impaired the exercise performance of mice. Mechanistically, OAA increased the c-Jun N-terminal kinase (JNK) phosphorylation and uncoupling protein 2 (UCP2) levels in skeletal muscle, which led to decreased energy substrate and enhanced glycolysis. On the other hand, OAA boosted muscle mitochondrial oxidative phosphorylation uncoupled with energy production. In addition, either UCP2 knockout or JNK inhibition totally reversed the effects of OAA on skeletal muscle. Therein, JNK mediated UCP2 activation with OAA-treated. Our studies reveal a novel role of OAA in skeletal muscle metabolism, which would shed light on the mechanism of muscle fatigue and weakness.

Anti-Obesity Effects of GABA in C57BL/6J Mice with High-Fat Diet-Induced Obesity and 3T3-L1 Adipocytes.

Obesity is the excessive accumulation of body fat resulting from impairment in energy balance mechanisms. In this study, we aimed to investigate the mechanism whereby GABA (γ-aminobutyric acid) prevents high-fat diet-induced obesity, and whether it induces lipolysis and browning in white adipose tissue (WAT), using high-fat diet (HFD)-fed obese mice and 3T3-L1 adipocytes. We demonstrated that GABA substantially inhibits the body mass gain of mice by suppressing adipogenesis and lipogenesis. Consistent with this result, histological analysis of WAT demonstrated that GABA decreases adipocyte size. Moreover, we show that GABA administration decreases fasting blood glucose and improves serum lipid profiles and hepatic lipogenesis in HFD-fed obese mice. Furthermore, Western blot and immunofluorescence analyses showed that GABA activates protein kinase A (PKA) signaling pathways that increase lipolysis and promote uncoupling protein 1 (UCP1)-mediated WAT browning. Overall, these results suggest that GABA exerts an anti-obesity effect via the regulation of lipid metabolism.

UCP2 promotes NSCLC proliferation and glycolysis via the mTOR/HIF-1α signaling.

Metabolic disturbance is a hallmark of cancers. Targeting key metabolic pathways and metabolism-related molecular could be a potential therapeutic approach. Uncoupling protein 2 (UCP2) plays a pivotal part in the malignancy of cancer and its capacity to develop resistance to pharmaceutical interventions. However, it is unclear about the mechanism of how UCP2 acts in the tumor growth and metabolic reprogramming process in non-small cell lung cancer (NSCLC). Here, we conducted qRT-PCR to investigate the expression of UCP2 in both NSCLC tissues and cell lines. Subsequent functional studies including colony formation assay, CCK-8 assay, and glycolysis assay were conducted to investigate the functions of UCP2 in NSCLC. The regulatory mechanism of UCP2 toward the mammalian target of rapamycin (mTOR) and hypoxia-inducible factor-1 alpha (HIF-1α) signaling in NSCLC was confirmed through western blotting. We observed a significant upregulation of UCP2 in both NSCLC tissues and cell lines. The increased expression of UCP2 has a strong association with a worse outlook. Silencing UCP2 remarkably dampened NSCLC cell proliferation and glycolysis capacities. Mechanically, UCP2 promoted NSCLC tumorigenesis partially via regulating the mTOR/HIF-1α axis. Taken together, we explored the functions as well as the mechanisms of the UCP2/mTOR/HIF-1α axis in NSCLC progression, uncovering potential biological signatures and targets for NSCLC treatment.

Genetic variation at the uncoupling protein 1, 2 and 3 loci and the response to long-term overfeeding

Genetic variation at the uncoupling protein 1, 2 and 3 loci and the response to long-term overfeeding

Expression, purification and folding of native like mitochondrial carrier proteins in lipid membranes

Mitochondrial Carrier Family proteins (MCFs) are located in the mitochondrial inner membrane and play essential roles in various cellular processes. Due to the relatively low abundance of many members of the family, in vitro structure and function determination of most MCFs require over-expression and purification of recombinant versions of these proteins. In this study, we report on a new method for overexpression of MCFs in Escherichia coli (E. coli) membranes, efficient purification of native-like proteins, and their reconstitution in mitochondrial inner membrane lipid mimics. cDNAs of Uncoupling Protein 4 (UCP4), Adenine Nucleotide Translocase (ANT) and Phosphate Translocase (PiT) were subcloned into the pET26b (+) expression vector such that fusion proteins with a short N-terminal pelB leader sequence and a six-histidine tag were produced to target the proteins toward the inner membrane of E. coli and facilitate affinity purification, respectively. Utilizing a modified autoinduction method, these proteins were overexpressed and extracted from the membrane of E. coli BL21 (DE3) and two modified strains, E. coli BL21 C43 (DE3) and E. coli BL21 Lobstr (DE3), in high yields. The proteins were then purified by immobilized metal affinity chromatography as monomers. Purity, identity, and concentration of the eluted monomers were determined by semi-native SDS-PAGE, Western blotting and mass spectrometry, and a modified Lowry assay, respectively. Cleavage of the pelB leader sequence from proteins was verified by mass spectrometric analysis. The purified proteins, surrounded by a shell of bacterial membrane lipids, were then reconstituted from the mild non-denaturing octyl glucoside (OG) detergent into phospholipid liposomes. Monomeric UCP4 spontaneously self-associated to form stable tetramers in lipid membranes, which is consistent with our previous studies. However, PiT and ANT remained dominantly monomeric in both detergent and liposome milieus, as detected by a combination of spectroscopic and electrophoretic methods. Native-like helical conformations of proteins were then confirmed by circular dichroism spectroscopy. Overall, this study demonstrates that targeting mitochondrial carrier family proteins to E. coli membranes provides an effective expression system for producing this family of proteins for biophysical studies.

LIGHT/TNFSF14 Affects Adipose Tissue Phenotype.

LIGHT/TNFSF14 is linked to several signaling pathways as a crucial member of a larger immunoregulatory network. It is primarily expressed in inflammatory effector cells, and high levels of LIGHT have been reported in obesity. Thus, with the aim of deepening the knowledge of the role of LIGHT on adipose tissue phenotype, we studied wild-type (WT), Tnfsf14-/-, Rag-/- and Rag-/Tnfsf14- (DKO) mice fed a normal diet (ND) or high-fat diet (HFD). Our results show that, although there is no significant weight gain between the mice with different genotypes, it is significant within each of them. We also detected an increase in visceral White Adipose Tissue (vWAT) weight in all mice fed HFD, together with the lowest levels of vWAT weight in Tnfsf14-/- and DKO mice fed ND with respect to the other strain. Inguinal WAT (iWAT) weight is significantly affected by genotype and HFD. The least amount of iWAT was detected in DKO mice fed ND. Histological analysis of vWAT showed that both the genotype and the diet significantly affect the adipocyte area, whereas the number is affected only by the genotype. In iWAT, the genotype and the diet significantly affect mean adipocyte area and number; interestingly, the area with the least adipocyte was detected in DKO mice fed ND, suggesting a potential browning effect due to the simultaneous lack of mature lymphocytes and LIGHT. Consistently, Uncoupling Protein 1 (UCP1) staining of iWAT demonstrated that few positive brown adipocytes appeared in DKO mice. Furthermore, LIGHT deficiency is associated with greater levels of UCP1, highlighting the lack of its expression in Rag-/- mice. Liver examination showed that all mice fed HFD had a steatotic liver, but it was particularly evident for DKO mice. In conclusion, our study demonstrates that the adipose tissue phenotype is affected by LIGHT levels but also much more by mature lymphocytes.

Postnatal growth restriction alters myocardial mitochondrial energetics in mice.

Postnatal growth restriction (PGR) can increase the risk of cardiovascular disease (CVD) potentially due to impairments in oxidative phosphorylation (OxPhos) within cardiomyocyte mitochondria. The purpose of this investigation was to determine if PGR impairs cardiac metabolism, specifically OxPhos. FVB (Friend Virus B-type) mice were fed a normal-protein (NP: 20% protein), or low-protein (LP: 8% protein) isocaloric diet 2weeks before mating. LP dams produce ∼20% less milk, and pups nursed by LP dams experience reduced growth into adulthood as compared to pups nursed by NP dams. At birth (PN1), pups born to dams fed the NP diet were transferred to LP dams (PGR group) or a different NP dam (control group: CON). At weaning (PN21), all mice were fed the NP diet. At PN22 and PN80, mitochondria were isolated for respirometry (oxygen consumption rate, ) and fluorimetry (reactive oxygen species emission, ) analysis measured as baseline respiration (LEAK) and with saturating ADP (OxPhos). Western blotting at PN22 and PN80 determined protein abundance of uncoupling protein 3, peroxiredoxin-6, voltage-dependent anion channel and adenine nucleotide translocator 1 to provide further insight into mitochondrial function. ANOVAs with the main effects of diet, sex and age with α-level of 0.05 was set a priori. Overall, PGR (7.8±1.1) had significant (P=0.01) reductions in respiratory control in complex I when compared to CON (8.9±1.0). In general, our results show that PGR led to higher electron leakage in the form of free radical production and reactive oxygen species emission. No significant diet effects were found in protein abundance. The observed reduced respiratory control and increased ROS emission in PGR mice may increase risk for CVD in mice.

UCP2-SIRT3 Signaling Relieved Hyperglycemia-Induced Oxidative Stress and Senescence in Diabetic Retinopathy.

Diabetic retinopathy (DR) is one of the most common reasons for blindness. uncoupling protein 2 (UCP2), an uncoupling protein located in mitochondria, has been reported to be related to metabolic and vascular diseases. This research aimed to illustrate the function and mechanism of UCP2 in the pathogenesis of DR. Human epiretinal membranes were collected to investigate the expression of UCP2 by quantitative real-time polymerase chain reaction (qRT-PCR) and immunofluorescence. Primary human retinal microvascular endothelial cells (HRECs) were cultured in high glucose (HG) to establish an in vitro cell model for DR. Flow cytometry analysis was used to measure intracellular reactive oxygen species (ROS). Senescence levels were evaluated by the senescence-associated beta-galactosidase (SA-β-gal) assay, the expression of senescence marker P21, and cell-cycle analysis. Adenovirus-mediated UCP2 overexpression or knockdown and specific inhibitors were administered to investigate the underlying regulatory mechanism. Proliferative fibrovascular membranes from patients with DR illustrated the downregulation of UCP2 and sirtuin 3 (SIRT3) by qRT-PCR and immunofluorescence. Persistent hyperglycemia-induced UCP2 downregulation in the progress of DR and adenovirus-mediated UCP2 overexpression protected endothelial cells from hyperglycemia-induced oxidative stress and senescence. Under hyperglycemic conditions, UCP2 overexpression attenuated NAD+ downregulation; hence, it promoted the expression and activity of SIRT3, an NAD+-dependent deacetylase regulating mitochondrial function. 3-TYP, a selective SIRT3 inhibitor, abolished the UCP2-mediated protective effect against oxidative stress and senescence. UCP2 overexpression relieved oxidative stress and senescence based on a novel mechanism whereby UCP2 can regulate the NAD+-SIRT3 axis. Targeting oxidative stress and senescence amelioration, UCP2-SIRT3 signaling may serve as a method for the prevention and treatment of DR and other diabetic vascular diseases.

Lactate coordinated with exercise promoted the browning of inguinal white adipose tissue.

Lactate, an important exercise metabolite, induces white adipose tissue browning by upregulated uncoupling protein 1 (UCP1) expression. However, the function of lactate during browning of inguinal white adipose tissue (iWAT) caused by exercise is unclear. Here, we considered lactate as an exercise supplement and investigated the effects of chronic pre-exercise lactate administration on energy metabolism and adipose tissue browning. C57B/L6 male mice (5 weeks of age) were divided into six groups. We evaluated the changes in blood lactate levels in each group of mice after the intervention. Energy expenditure was measured after the intervention immediately by indirect calorimetry. The marker protein levels and gene expressions were determined by western-blot and quantitative real-time PCR. HIIT significantly decreased adipose tissue weight while increased energy expenditure and the expression of UCP1 in iWAT; however, these regulations were inhibited in the DCA+HIIT group. Compared with the MICT and LAC groups, long-term lactate injection before MICT led to lower WAT weight to body weight ratios and higher energy expenditure in mice. Furthermore, the marker genes of browning in iWAT, such as Ucp1 and Pparγ, were significantly increased in the LAC+MICT group than in the other groups, and the expression of monocarboxylate transporter-1 (Mct1) mRNA was also significantly increased. Lactate was involved in exercise-mediated browning of iWAT, and its mechanism might be the increased of lactate transport through MCT1 or PPARγ upregulation induced by exercise. These findings suggest exogenous lactate may be a new exercise supplement to regulate metabolism.

Dysregulation of mitochondrial dynamics mediated aortic perivascular adipose tissue-associated vascular reactivity impairment under excessive fructose intake

Excessive fructose intake presents the major risk factor for metabolic cardiovascular disease. Perivascular adipose tissue (PVAT) is a metabolic tissue and possesses a paracrine function in regulating aortic reactivity. However, whether and how PVAT alters vascular function under fructose overconsumption remains largely unknown. In this study, male Sprague-Dawley rats (8 weeks old) were fed a 60% high fructose diet (HFD) for 12 weeks. Fasting blood sugar, insulin, and triglycerides were significantly increased by HFD intake. Plasma adiponectin was significantly enhanced in the HFD group. The expression of uncoupling protein 1 (UCP1) and mitochondrial mass were reduced in the aortic PVAT of the HFD group. Concurrently, the expression of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) and mitochondrial transcription factor A (TFAM) were suppressed. Furthermore, decreased fusion proteins (OPA1, MFN1, and MFN2) were accompanied by increased fission proteins (FIS1 and phospho-DRP1). Notably, the upregulated α-smooth muscle actin (α-SMA) and osteocalcin in the PVAT were concurrent with the impaired reactivity of aortic contraction and relaxation. Coenzyme Q10 (Q, 10 mg/100 mL, 4 weeks) effectively reversed the aforementioned events induced by HFD. Together, these results suggested that the dysregulation of mitochondrial dynamics mediated HFD-triggered PVAT whitening to impair aortic reactivity. Fortunately, coenzyme Q10 treatment reversed HFD-induced PVAT whitening and aortic reactivity.

Ketogenic diet with aerobic exercise can induce fat browning: potential roles of β-hydroxybutyrate.

Despite evidence suggesting that metabolic intermediates like β-HB influence white adipose tissue (WAT) metabolism, the precise molecular mechanisms remain unclear. The aim of this study was to investigate the impact of beta-hydroxybutyrate (β-HB) on the fat browning program and to explore the underlying molecular mechanisms using both in vitro and in vivo models. We assessed the effects of β-HB on fat browning in adipocytes using 3T3-L1 cells and rat models. We evaluated the effects of β-HB on fat browning, thermogenesis, lipid accumulation, adipokine expression, and mitochondrial biogenesis by treating mature 3T3-L1 adipocytes with sodium β-HB for 24 h or by continuously exposing preadipocytes to β-HB during the 8-day differentiation process. Male Sprague Dawley rats were divided into control, exercise only (EX), ketogenic diet only (KD), and combined exercise and ketogenic diet (KE) groups for an 8-week intervention involving diet and/or exercise. After intervention, we evaluated WAT histology, plasma lipids and adipokines, and the expression of markers related to fat browning, thermogenesis and mitochondrial biogenesis in WAT of rats. In our adipocyte culture experiments, β-HB reduced intracellular lipid accumulation by enhancing lipolysis and stimulated the expression of thermogenic and fat browning genes like uncoupling protein 1 (UCP1), PR domain containing 16 (PRDM16), and adipokines such as fibroblast growth factor 21 (FGF21) and Fibronectin type III domain-containing protein 5 (FDNC5). Additionally, β-HB activated the AMPK-SIRT1-PGC-1α pathway, with UCP1 and PRDM16 upregulation mediated by β-HB intracellular action and SIRT1 activity. In animal experiments, KE group raised β-HB levels, decreasing body weight and blood lipids. KD with EX promoted WAT browning possibly via AMPK-SIRT1-PGC-1α, augmenting PRDM16, UCP1, FGF21, and FNDC5 expression. β-HB induction via KD and/or EX shows potential in promoting WAT browning by activating mitochondrial biogenesis, lipolysis, and thermogenesis, suggesting that dietary and physical intervention inducing β-HB may benefit metabolic health.

Transcutaneous carbon dioxide suppresses skeletal muscle atrophy in a mouse model of oral squamous cell carcinoma.

Cancer cachexia causes skeletal muscle atrophy, impacting the treatment and prognosis of patients with advanced cancer, but no treatment has yet been established to control cancer cachexia. We demonstrated that transcutaneous application of carbon dioxide (CO2) could improve local blood flow and reduce skeletal muscle atrophy in a fracture model. However, the effects of transcutaneous application of CO2 in cancer-bearing conditions are not yet known. In this study, we calculated fat-free body mass (FFM), defined as the skeletal muscle mass, and evaluated the expression of muscle atrophy markers and uncoupling protein markers as well as the cross-sectional area (CSA) to investigate whether transcutaneous application of CO2 to skeletal muscle could suppress skeletal muscle atrophy in cancer-bearing mice. Human oral squamous cell carcinoma was transplanted subcutaneously into the upper dorsal region of nude mice, and 1 week later, CO2 gas was applied to the legs twice a week for 4 weeks and FFM was calculated by bioimpedance spectroscopy. After the experiment concluded, the quadriceps were extracted, and muscle atrophy markers (muscle atrophy F-box protein (MAFbx), muscle RING-finger protein 1 (MuRF-1)) and uncoupling protein markers (uncoupling protein 2 (UCP2) and uncoupling protein 3 (UCP3)) were evaluated by real-time polymerase chain reaction and immunohistochemical staining, and CSA by hematoxylin and eosin staining. The CO2-treated group exhibited significant mRNA and protein expression inhibition of the four markers. Furthermore, immunohistochemical staining showed decreased MAFbx, MuRF-1, UCP2, and UCP3 in the CO2-treated group. In fact, the CSA in hematoxylin and eosin staining and the FFM revealed significant suppression of skeletal muscle atrophy in the CO2-treated group. We suggest that transcutaneous application of CO2 to skeletal muscle suppresses skeletal muscle atrophy in a mouse model of oral squamous cell carcinoma.

Effect of Oleuropein on Anti-Obesity and Uncoupling Protein 1 Level in Brown Adipose Tissue in Mild Treadmill Walking Rats with Diet-Induced Obesity.

Oleuropein aglycone (OA), which is the absorbed form of oleuropein, is a major phenolic compound in extra virgin olive oil. We analyzed the anti-obesity effect of OA intake combined with mild treadmill walking (MTW, 4 m/min for 20 min/d, 5-6 d/wk, without electric shocks and slope) in rats under a high-fat diet (HF). Four-week-old male Sprague-Dawley rats (n=28) were equally divided into four groups: control (HF), 0.08% oleuropein-supplemented HF (HFO), HF with MTW (HF+W), and HFO with MTW (HFO+W) groups. After 28 d, the inguinal subcutaneous fat content and weight gain were significantly lower in the HFO+W group than in the control group. The HFO+W group also had significantly higher levels of urinary noradrenaline secretion, interscapular brown adipose tissue, uncoupling protein 1, brain transient receptor potential ankyrin subtype 1 (TRPA1), vanilloid subtype 1 (TRPV1), and brain-derived neurotrophic factor (BDNF) than the control group. Especially, the HFO+W group showed a synergistic effect on noradrenaline secretion. Therefore, OA combined with MTW may accelerate the enhancement of UCP1 and BDNF levels in rats with HF-induced obesity by increasing noradrenaline secretion after TRPA1 and TRPV1 activation.

Differential effects of milk, yogurt, and cheese on energy homeostasis and brown adipose tissue phenotype in high-fat diet-induced obese mice.

Aim: We hypothesized that milk, yogurt, and cheese have differential impacts on energy expenditure (EE) and obesity in mice fed a high-fat diet (HFD). Methods: C57BL/6 mice (n = 16 per group) were fed a HFD or a HFD supplemented with fat-free milk (MILK), fat-free plain yogurt (YOG), or reduced-fat cheddar cheese (CHE; 19 kcal% fat), each provided at 10% of the daily energy intake, for 8 weeks. EE was quantified using a metabolic chamber. Metabolic pathways related to BAT mitochondrial function and uncoupling protein 1 (UCP1) abundance were assessed. Serum lipidomic profiles were analyzed to identify potential mediators of the observed effects. Results: MILK supplementation lowered weight gain and fat accumulation and enhanced EE and BAT thermogenesis, perhaps via the SIRT1-AMPK-PGC1α axis in BAT. This led to elevated UCP1 abundance and enhanced the abundance of hormone-sensitive lipase (HSL). MILK also altered serum lipid species, indicating enhanced energy use, and promoted BAT thermogenesis and mitochondrial function pathways. YOG exhibited a similar pattern but a lower magnitude of effects than MILK on reducing weight gain and fat mass, increasing EE, and BAT thermogenic proteins, including AMPK-PGC1α-UCP1. Both MILK and YOG showed a relative increase in serum PC 15:0_15:0 and LPC 15:0. In contrast, CHE reduced weight gain and increased EE without impacting BAT thermogenesis proteins or serum lipid species. Conclusion: Our study showed that MILK, YOG, and CHE reduced weight gain in mice on a HFD by increasing EE. MILK and YOG also up-regulated BAT thermogenesis, while both additionally altered lipids involved in fat metabolism and inflammation. CHE did not affect BAT thermogenesis and lipid species compared to HFD.

Overexpression of TRPV6 Inhibits Coronary Atherosclerosis–Related Inflammatory Response and Cell Apoptosis via the PKA/UCP2 Pathway

Objective: This research is aimed at unravelling the intricate relationship between transient receptor potential vanilloid 6 (TRPV6), protein kinase A (PKA), uncoupling protein 2 (UCP2), and atherosclerosis. By shedding light on the role of the TRPV6/PKA/UCP2 pathway in inhibiting inflammatory response and cell apoptosis in coronary atherosclerotic plaques, this study provides valuable insights into potential therapeutic targets for treating coronary artery disease (CAD).Methods: We established animal and cell models of atherosclerosis. The expression of TRPV6 was measured using immunohistochemistry and immunofluorescence. Cytokine levels were detected by enzyme‐linked immunosorbent assay (ELISA). Cell viability and apoptosis ratio were measured using cell counting kit‐8 (CCK‐8) and flow cytometry. The binding relationship between TRPV6 and PKA was validated using chromatin immunoprecipitation (CHIP) and coimmunoprecipitation (CoIP). Finally, the expression of the TRPV6/PKA/UCP2 signaling pathway and apoptosis‐related factors was detected using western blot (WB) and quantitative real‐time polymerase chain reaction (qRT‐PCR).Results: TRPV6 was significantly decreased in atherosclerosis mouse and cell model. CHIP and CoIP assays indicated that TRPV6 binds to PKA and positively regulated its expression in oxidized low‐density lipoprotein (ox‐LDL)–treated human umbilical vein endothelial cells (HUVECs). Overexpression of TRPV6 significantly increased cell viability and inhibited apoptosis, whereas silencing TRPV6 had the opposite effect. Additionally, the overexpression of TRPV6 remarkably declined the expression of tumor necrosis factor‐alpha (TNF‐α), interleukin‐6 (IL‐6), and interleukin‐1 beta (IL‐1β). However, after silencing PKA, this effect was partially reversed, the cell viability and inflammatory response remarkably enhanced, and apoptosis significantly declined in oe‐TRPV6 + si‐PKA group.Conclusions: In summary, our study demonstrated that TRPV6 inhibited apoptosis and inflammatory response in the atherosclerosis cell model through the regulation of the PKA/UCP2 pathway.

The futile creatine cycle and the synthesis of fatty acids in inguinal white adipose tissue from growing rats, submitted to a hypoprotein-hyperglycidic diet for 15 days.

The low-protein, high-carbohydrate (LPHC) diet administered to growing rats soon after weaning, for 15 days, promoted an increase in energy expenditure by uncoupling protein 1 (UCP1) in interscapular brown adipose tissue, and also due to the occurrence of the browning process in the perirenal white adipose tissue (periWAT). However, we believe that inguinal white adipose tissue (ingWAT) may also contribute to energy expenditure through other mechanisms. Therefore, the aim of this work is to investigate the presence of the futile creatine cycle, and the origin of lipids in ingWAT, since that tissue showed an increase in the lipids content in rats submitted to the LPHC diet for 15 days. We observed increases in creatine kinase and alkaline phosphatase activity in ingWAT, of the LPHC animals. The mitochondrial Nicotinamide adenine dinucleotide reduced/nicotinamide adenine dinucleotide oxidized ratio is lower in ingWAT of LPHC animals. In the LPHC animals treated with β-guanidinopropionic acid, the extracellular uptake of creatine in ingWAT was lower, as was the rectal temperature. Regarding lipid metabolism, we observed that in ingWAT, lipolysis in vitro when stimulated with noradrenaline is lower, and there were no changes in baseline levels. In addition, increases in the activity of enzymes were also observed: malic, glucose-6-phosphate dehydrogenase, and ATP-citrate lyase, in addition to an increase in the PPARγ content. The results show the occurrence of the futile creatine cycle in ingWAT, and that the increase in the relative mass may be due to an increase in de novo fatty acid synthesis.

Unveiling the potential of marine-derived diterpenes from the order Alcyonacea as promising anti-obesity agents

Metabolic syndrome (MS) represents a global health challenge characterized by various metabolic disorders, including HOMA-IR (insulin resistance), obesity, dyslipidemia, and hypertension. In our pursuit of identifying natural alternatives for the development of effective and safe anti-obesity medications, we examined the potential of the methanolic extract of the Red Sea derived soft coral Sarcophyton glaucum, where serum levels of glucose, insulin, HOMA-IR, lipid profile, fetuin A and B, PTP1Β (Protein tyrosine phosphatase 1B), adropin and omentin were determined. Furthermore, the expression of the UCP1 (Uncoupling protein 1) and PPARGC1A (Peroxisome proliferator–activated receptor-g coactivator-1a) genes have been assessed, to evaluate the anti-obesity potential of S. glaucum organic extract. Our findings demonstrated a significant decrease in glucose, HOMA-IR, cholesterol, triglyceride, LDL-C, fetuin A and B, and PTP1Β levels, accompanied by a significant increase in insulin, HDL-C, adropin, omentin, UCP1, and PPARGC1A expression after treatment with the soft coral extract. These promising outcomes can be attributed to the remarkable ingredients present in the extract, which were further supported by histopathological findings. In addition, a virtual screening protocol including molecular docking (MDock) and Structure-Activity Relationships (SARs) of 27 marine diterpenes was also explored to identify potential PTP1Β inhibitors targeting simultaneously the catalytic site and allosteric site, as well as fetuin A modulators. Moreover, the six most promising predicted marine diterpenes (4, 8, 9, 10, 13 and 14) were investigated for their pharmacokinetic properties, druglike nature and medicinal chemistry friendliness using the SwissADME platform. Of these, four marine diterpenes (4, 8, 9, and 10) were predicted to exhibit the appropriate drug-like properties.

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

Early Adipogenesis and Upregulation of UCP1 in Mesenchymal Stromal Cells Stimulated by Devitalized Microfragmented Fat (MiFAT).

Adipose tissue is mainly composed by adipocytes. Moreover, mesenchymal stromal/stem cells (MSCs), macrophages, endothelial cells, and extracellular matrix components are present. The variety of molecules as cytokines and growth factors of its structure very rich in blood vessel makes it also similar to a true endocrine organ that however needs still to be fully investigated. In our study, we used human lipoaspirate to obtain mechanically microfragmented fat (MiFAT) which was washed and then devitalized by freezing-thawing cycles. In our experiments, thawed MiFAT was used to stimulate cultures of MSCs from two different sources (adipose tissue and gingiva papilla) in comparison with a traditional stimulation in vitro obtained by culturing MSCs with adipogenic medium. MSCs stimulated with MiFAT showed a very early production of lipid droplets, after only 3 days, that correlated with an increased expression of adipokines. Furthermore, a significant upregulation of PPAR gamma 1 alpha coactivator (PPARGC1A) was observed with an overexpression of uncoupling protein 1 (UCP1) that suggest a pattern of differentiation compatible with the beige-brown fat.