Glycyrrhizin (Glycyrrhizic Acid) HMGB1 (high mobility group box 1) inhibitor upregulate mitochondrial function in adipocyte,cell viability and in-silico study

BackgroundMitochondrial plays a vital role in regulating obesity and related comorbidity. Targeting mitochondrial function could be a potent therapeutic approach to inhibit metabolic-related diseases like obesity, liver disease. Prolonged use of existing drug moieties demonstrated severe adverse effects.MethodsWe apply Ucp1-A-GFP immortalized reporter cell lines and HEK293T cell lines to evaluate cell viability, mitochondrial ATP production, and the in-silico model.ResultsWe found Glycyrrhizin, an HMGB1 (high mobility group box 1) inhibitor, plays a significant role in modulating mitochondrial function against obesity. At the cellular level, the adipocytes treated with Glycyrrhizin have increased mitochondrial function. Further analysis shows that compared with the control group, the cells in the treatment group contain more mitochondria. Glycyrrhizin demonstrated a nontoxic effect on the HEK293T cell line, upregulating mitochondrial DNA and reducing mitochondrial ATP production levels. In-silico study exhibited drug-protein interaction and binding side with UCP1.ConclusionGlycyrrhizin improves mitochondrial function that would be an effective drug candidate to treat metabolic diseases and obesity-related diseases. Further investigation will require both the human and animal models to reveal new insight into the mechanism against obesity, metabolic diseases or mitochondrial dysfunction-related diseases.     

Epigenetic drug (XL019) JAK2 inhibitor increases mitochondrial function in brown adipocytes by upregulating mitochondrial uncoupling protein 1 (UCP1), screening of epigenetic drug libraries,cell viability,and in-silico studies

BackgroundAt present lacking of effective and safe anti-obesity drugs available leads to initiate obesity worldwide that promotes several diseases like cardiovascular diseases, liver diseases, and NASH. The development of new therapeutics is an emergency demand to cure obesity-related diseases. Mitochondrial uncoupling protein 1 (UCP1) gene could be a potential target to develop new drug moieties that can treat obesity-related diseases.MethodsWe used a GFP reporter cell line to screen epigenetic drug libraries to identify UCP1 regulators that could be effective drug candidates to treat obesity-related diseases. In this study, we employed an in-silico study that revealed drug-protein interaction and stability of drugs with protein.ResultsScreening epigenetic drug libraries, we identified XL019 significant TYK2, JAK2, and JAK3, inhibitors that can significantly promote UCP1 gene expression in brown adipocytes. Here, we found that XL019 plays a vital role to modulates mitochondrial function and could be beneficial against obesity. Further analysis shows that XL019 significantly improved mitochondrial ATP production and mitochondrial DNA copy number of adipocytes compared with the control group. The in-silico study demonstrated drug-protein interaction and binding side with UCP1 gene. Thus XL019 improves mitochondrial function that would be effective drug candidate to treat metabolic diseases and obesity-related diseases.ConclusionIn this study, we confirm the potential effect of the XL019 epigenetic drug that modulates mitochondrial function and in-silico study on drug-likeness, stability, and safety profile. Further investigation will reveal the new insight into the mechanism of action against obesity, metabolic diseases ( NASH, Fibrosis, cardiac diseases and so on), by modulation of the mitochondrial UCP1 gene and mitochondrial function.     

Zebrafish and Flavonoids: Adjuvants against Obesity

Obesity is a pathological condition, defined as an excessive accumulation of fat, primarily caused by an energy imbalance. The storage of excess energy in the form of triglycerides within the adipocyte leads to lipotoxicity and promotes the phenotypic switch in the M1/M2 macrophage. These changes induce the development of a chronic state of low-grade inflammation, subsequently generating obesity-related complications, commonly known as metabolic syndromes. Over the past decade, obesity has been studied in many animal models. However, due to its competitive aspects and unique characteristics, the use of zebrafish has begun to gain traction in experimental obesity research. To counteract obesity and its related comorbidities, several natural substances have been studied. One of those natural substances reported to have substantial biological effects on obesity are flavonoids. This review summarizes the results of studies that examined the effects of flavonoids on obesity and related diseases and the emergence of zebrafish as a model of diet-induced obesity.     

Buddleja officinalis Maximowicz extract inhibits lipid accumulation on adipocyte differentiation in 3T3-L1 cells and high-fat mice

Obesity is a global health problem. It is also known to be a risk factor for the development of metabolic disorders, type 2 diabetes, systemic hypertension, cardiovascular disease, dyslipidemia, and atherosclerosis. In this study, we elucidated that Buddleja officinalis Maximowicz extract significantly inhibited lipid accumulation during 3T3-L1 adipocyte differentiation. Furthermore, Buddleja officinalis Maximowicz extract reduced the body weight gain induced through feeding a high-fat diet to C57BL/6 mice. The treatment of Buddleja officinalis Maximowicz extract significantly reduced the adipose tissue weight to 2.7/100 g of body weight in high-fat mice. When their adipose tissue morphology was investigated for histochemical staining, the distribution of cell size in the high-fat diet groups was hypertrophied compared with those from Buddleja officinalis Maximowicz extract-treated mice. In addition, in Buddleja officinalis Maximowicz extract-treated mice, a significant reduction of serum triglyceride and T-cholesterol was observed at to 21% and 17%, respectively. The discovery of bioactive compounds from diet or dietary supplementation is one of possible ways to control obesity and to prevent or reduce the risks of various obesity-related diseases. These results support that Buddleja officinalis Maximowicz extract is expected to create the therapeutic interest with respect to the treatment of obesity.     

Association of anti-obesity activity of N-acetylcysteine with metallothionein-II down-regulation

People with upper body or visceral obesity have a much higher risk of morbidity and mortality from obesity-related metabolic disorders than those with lower body obesity. In an attempt to develop therapeutic strategies targeting visceral obesity, depot- specific differences in the expression of genes in omental and subcutaneous adipose tissues were investigated by DNA array technology, and their roles in adipocyte differentiation were further examined. We found that levels of metallothionein-II (MT-II) mRNA and protein expression were higher in omental than in subcutaneous adipose tissues. The study demonstrates that MT-II may play an important role in adipocyte differentiation of 3T3L1 preadipocytes, and that N-acetylcysteine (NAC) inhibits the adipocyte differentiation of 3T3L1 cells by repressing MT-II in a time- and dose-dependent manner. Furthermore, the intraperitoneal administration of NAC to rats and mice resulted in a reduction of body weights, and a marked reduction in visceral fat tissues. These results suggest that MT-II plays important roles in adipogenesis, and that NAC may be useful as an anti-obesity drug or supplement.     

Elecampane (Inula helenium) Root Extract and Its Major Sesquiterpene Lactone,Alantolactone, Inhibit Adipogenesis of 3T3-L1 Preadipocytes

Recent studies have shown that Nur77 and AMPKα play an important role in regulating adipogenesis and isoalantolactone (ISO) dual-targeting AMPKα and Nur77 inhibits adipogenesis. In this study, we hypothesized that Inula helenium (elecampane) root extract (IHE), which contains two sesquiterpene lactones, alantolactone (ALA) and ISO, as major compounds, might inhibit adipogenesis. Here, we found that ALA and IHE simultaneously target AMPKα and Nur77 and inhibited adipogenic differentiation of 3T3-L1 cells, accompanied by the decreased expression of adipocyte markers. Further mechanistic studies demonstrated that IHE shares similar mechanisms of action with ISO that reduce mitotic clonal expansion during the early phase of adipogenic differentiation and decrease expression of cell cycle regulators. These results suggest that IHE inhibits adipogenesis, in part, through co-regulation of AMPKα and Nur77, and has potential as a therapeutic option for obesity and related metabolic dysfunction.     

Delivery of Thyronamines (TAMs) to the Brain: A Preliminary Study

Recent reports highlighted the significant neuroprotective effects of thyronamines (TAMs), a class of endogenous thyroid hormone derivatives. In particular, 3-iodothyronamine (T1AM) has been shown to play a pleiotropic role in neurodegeneration by modulating energy metabolism and neurological functions in mice. However, the pharmacological response to T1AM might be influenced by tissue metabolism, which is known to convert T1AM into its catabolite 3-iodothyroacetic acid (TA1). Currently, several research groups are investigating the pharmacological effects of T1AM systemic administration in the search of novel therapeutic approaches for the treatment of interlinked pathologies, such as metabolic and neurodegenerative diseases (NDDs). A critical aspect in the development of new drugs for NDDs is to know their distribution in the brain, which is fundamentally related to their ability to cross the blood–brain barrier (BBB). To this end, in the present study we used the immortalized mouse brain endothelial cell line bEnd.3 to develop an in vitro model of BBB and evaluate T1AM and TA1 permeability. Both drugs, administered at 1 µM dose, were assayed by high-performance liquid chromatography coupled to mass spectrometry. Our results indicate that T1AM is able to efficiently cross the BBB, whereas TA1 is almost completely devoid of this property.     

Further understanding of fat biology: Lessons from a fat fly

Obesity is a leading risk factor for insulin resistance, hypertension, hyperlipidemia, and cardiovascular complications, collectively referred to as metabolic diseases. Given the prevalence of obesity and its associated medical problems, new strategies are required to prevent or treat obesity and obesity-related metabolic effects. Here we summarize contributors of obesity, and molecular mechanisms controlling adipogenesis from studies in mammalian systems. We also discuss the possibilities of using Drosophila as a genetic model system to advance our understanding of players in fat biology.     

Isoliquiritigenin Enhances the Beige Adipocyte Potential of Adipose-Derived Stem Cells by JNK Inhibition

Human adipose-derived stem cells (hASCs) can be isolated from fat tissue and have attracted interest for their potential therapeutic applications in metabolic disease. hASCs can be induced to undergo adipogenic differentiation in vitro by exposure to chemical agents or inductive growth factors. We investigated the effects and mechanism of differentiating hASC-derived white adipocytes into functional beige and brown adipocytes with isoliquiritigenin (ILG) treatment. Here, we showed that hASC-derived white adipocytes could promote brown adipogenesis by expressing both uncoupling protein 1 (UCP1) and PR/SET Domain 16 (PRDM16) following low-dose ILG treatments. ILG treatment of white adipocytes enhanced the expression of brown fat-specific markers, while the expression levels of c-Jun N-terminal kinase (JNK) signaling pathway proteins were downregulated. Furthermore, we showed that the inhibition of JNK phosphorylation contributed to white adipocyte differentiation into beige adipocytes, which was validated by the use of SP600125. We identified distinct regulatory effects of ILG dose responses and suggested that low-dose ILG induced the beige adipocyte potential of hASCs via JNK inhibition.     

Ferulic Acid Stimulates Adipocyte-Specific Secretory Proteins to Regulate Adipose Homeostasis in 3T3-L1 Adipocytes

Obesity has recently emerged as a public health issue facing developing countries in the world. It is caused by the accumulation of fat in adipose, characterized by insulin resistance, excessive lipid accumulation, inflammation, and oxidative stress, leading to an increase in adipokine levels. Herein, we investigated the capacity of a bioactive polyphenolic compound (ferulic acid (FA)) to control adipocyte dysfunction in 3T3-L1 adipocytes (in vitro). Key adipocyte differentiation markers, glycerol content, lipolysis-associated mRNA, and proteins were measured in experimental adipocytes. FA-treated adipocytes exhibited downregulated key adipocyte differentiation factors peroxisome proliferator-activated receptor-γ (PPAR-γ), CCAT enhancer binding-proteins—α (C/EBP-α) and its downstream targets in a time-dependent manner. The FA-treated 3T3-L1 adipocytes showed an increased release of glycerol content compared with non-treated adipocytes. Also, FA treatment significantly up-regulated the lipolysis-related factors, including p-HSL, and p-perilipin, and down-regulated ApoD, Sema3C, Cxcl12, Sfrp2, p-stearoyl-CoA desaturase 1 (SCD1), adiponectin, and Grk5. Also, the FA treatment showed significantly down-regulated adipokines leptin, chemerin, and irisin than the non-treated cells. The present findings indicated that FA showed significant anti-adipogenic and lipogenic activities by regulating key adipocyte factors and enzyme, enhanced lipolysis by HSL/perilipin cascade. FA is considered a potent molecule to prevent obesity and its associated metabolic changes in the future.     

Atrophy of brown adipocytes in the adult mouse causes transformation into white adipocyte-like cells

Adipose tissue is an important endocrine regulator of glucose metabolism and energy homeostasis. Researches have focused on this tissue not only as a target for pharmacotherapy of obesity and insulin resistance but also as an endocrine tissue with leptin secretion and high insulin sensitivity. Brown adipose tissue (BAT) additionally plays a unique role in thermoregulation through the mitochondrial uncoupling protein 1 (UCP1), which uncouples oxidative phosphorylation. As a genetic tissue ablation model of BAT, we made transgenic mice expressing herpes simplex virus thymidine kinase (HSV-TK) driven by the brown adipocyte- specific UCP1 minimal regulatory element. The HSV-TK transgene was expressed specifically in BAT and more than 35% increase of apoptosis was induced by ganciclovir (GCV) treatment. Nevertheless, the expression level was not high enough to induce BAT ablation in GCV-treated adult mice. Importantly, however, we found that brown adipocytes in the periphery of interscapular BAT were transformed into white adipocyte-like unilocular cells. These cells express white adipocyte-specific leptin protein but are different in the ultrastructure of mitochondria from classical white adipocytes. Our data indicates that atrophy of BAT causes transformation into white adipocyte-like cells in the adult mouse and also suggests that further molecular understanding of adipocyte plasticity using our transgenic mouse model might be beneficial for the development of anti-obesity/anti-diabetic therapies.     

Ribes fasciculatum Ameliorates High-Fat-Diet-Induced Obesity by Elevating Peripheral Thermogenic Signaling

Ribes fasciculatum has been consumed as a food and as a traditional medicine for treating autoimmune diseases and aging in diverse countries. A previous study showed that a mixture of Ribes fasciculatum and Cornus officinalis prohibited adipocyte differentiation and lipid accumulation in preadipocytes and suppressed diet-induced obesity. Nevertheless, the mechanism of R. fasciculatum to regulate energy homeostasis solely through thermogenic signaling remains unclear. Thus, we investigated its effects on energy homeostasis using R. fasciculatum fed to C57BL/6 mice with a 45% high-fat diet. Chronic consumption of R. fasciculatum decreased the body weight of obese mice with increasing food intakes and improved metabolic-syndrome-related phenotypes. Therefore, we further tested its thermogenic effects. Cold chamber experiments and qPCR studies indicated that R. fasciculatum elevated thermogenic signaling pathways, demonstrated by increased body temperature and uncoupling protein 1 (UCP1) signaling in the white and brown adipose tissues. Afzelin is one major known compound derived from R. fasciculatum. Hence, the isolated compound afzelin was treated with preadipocytes and brown adipocytes for cell viability and luciferase assay, respectively, to further examine its thermogenic effect. The studies showed that the response of afzelin was responsible for cell viability and the increased UCP1. In conclusion, our data indicated that R. fasciculatum elevated peripheral thermogenic signaling through increased UCP1 via afzelin activation and ameliorated diet-induced obesity.     

ER-associated CTRP1 regulates mitochondrial fission via interaction with DRP1

C1q/TNF-related protein 1 (CTRP1) is a CTRP family member that has collagenous and globular C1q-like domains. The secreted form of CTRP1 is known to be associated with cardiovascular and metabolic diseases, but its cellular roles have not yet been elucidated. Here, we showed that cytosolic CTRP1 localizes to the endoplasmic reticulum (ER) membrane and that knockout or depletion of CTRP1 leads to mitochondrial fission defects, as demonstrated by mitochondrial elongation. Mitochondrial fission events are known to occur through an interaction between mitochondria and the ER, but we do not know whether the ER and/or its associated proteins participate directly in the entire mitochondrial fission event. Interestingly, we herein showed that ablation of CTRP1 suppresses the recruitment of DRP1 to mitochondria and provided evidence suggesting that the ER–mitochondrion interaction is required for the proper regulation of mitochondrial morphology. We further report that CTRP1 inactivation-induced mitochondrial fission defects induce apoptotic resistance and neuronal degeneration, which are also associated with ablation of DRP1. These results demonstrate for the first time that cytosolic CTRP1 is an ER transmembrane protein that acts as a key regulator of mitochondrial fission, providing new insight into the etiology of metabolic and neurodegenerative disorders.Subject terms: Endoplasmic reticulum, Mitochondria     

A temperature-regulated bioorthogonal reaction to target lysine: Hemiacetal pharmacophore in genipin irreversibly binds with UCP2, inhibiting mitochondrial thermogenesis

Mitochondria are essential for eukaryotic life as powerhouses for energy metabolism. Excessive mitochondrial hyperthermia and reactive oxygen species (ROS) production have been associated with aging, cancer, neurodegenerative diseases, and other disorders. Uncoupling protein 2 (UCP2) is the effector responsible for regulating cellular thermogenesis and ROS production via dissipating protons in an electrochemical gradient. A UCP2 inhibitor named genipin (GNP) is being researched for its effect on mitochondrial temperature, but little is known about its mechanisms. This study developed several molecular probes to explore the interactions between GNP and UCP2. The result indicated that the hemiacetal structure in GNP could selectively react with the ɛ-amine of lysine on the UCP2 proton leakage channel through ring-opening condensation at the mitochondrial, cellular, and animal levels. A notable feature of the reaction is its temperature sensitivity and ability to conjugate with UCP2 at high fever as lysine-specific covalent inhibitors that prevent mitochondrial thermogenesis. The result not only clarifies the existence of an antipyretic properties of GNP via its irreversible coupling to UCP2, but also reveals a bioorthogonal reaction of hemiacetal iridoid aglycone for selectively binding with the ɛ-amine of lysine on proteins.     

Electrically wired mitochondrial electrodes for measuring mitochondrial function for drug screening

In the continual search of new therapeutics, many possible drug candidates are excluded, because they are found to negatively affect mitochondrial function. We have developed an approach for directly, electrochemically assaying mitochondrial metabolic activity as a function of metabolic substrate to determine drug toxicity. By wiring mouse mitochondria to a carbon electrode surface, electrons can be intercepted before they reach Complex IV, the terminal step of electron transport chain. The electrons are rerouted, to a separate electrode of the electrochemical cell, the cathode. This allows for the direct measurement of electrical current and potential of the mitochondria during their oxidation of substrates such as pyruvate and fatty acids when there are different concentrations of drug present. This analytical technique has been shown to reliably assay several classical mitochondrial toxins and exhibits potential for the further development of a drug candidate screening technique, as well as other applications where the quantitative study of mitochondrial dysfunction is important.     

Swim training improves leptin receptor deficiency-induced obesity and lipid disorder by activating uncoupling proteins

Leptin receptor deficiency causes morbid obesity and hyperlipidemia in mice. Since physical exercise enhances energy expenditure, it is an important part of successful weight-control regimens. We investigated the mechanism by which swim training regulates leptin receptor deficiency-induced obesity and lipid disorder in a mouse model of obesity (obese db/db mouse). Swim training for 6 weeks significantly decreased body weight gain and adipose tissue mass in both sexes of obese and lean mice, compared to their respective sedentary controls. These effects were particularly evident in obese mice. Swim training also caused significant decreases in serum levels of triglycerides, free fatty acids and total cholesterol in both obese and lean mice. In obese mice, swim training increased the levels of mRNAs and proteins encoding uncoupling protein 1 (UCP1), UCP2 and UCP3 in brown adipose tissue, white adipose tissue and skeletal muscle, respectively. In conclusion, these findings suggest that, in mice, swim training can effectively prevent body weight gain, adiposity and lipid disorders caused by leptin receptor deficiency, in part through activation of UCPs in adipose tissue and skeletal muscle, which may contribute to alleviating metabolic syndromes, such as obesity, hyperlipidemia and type 2 diabetes.     

SENP2 regulates mitochondrial function and insulin secretion in pancreatic β cells

Increasing evidence has shown that small ubiquitin-like modifier (SUMO) modification plays an important role in metabolic regulation. We previously demonstrated that SUMO-specific protease 2 (SENP2) is involved in lipid metabolism in skeletal muscle and adipogenesis. In this study, we investigated the function of SENP2 in pancreatic β cells by generating a β cell-specific knockout (Senp2-βKO) mouse model. Glucose tolerance and insulin secretion were significantly impaired in the Senp2-βKO mice. In addition, glucose-stimulated insulin secretion (GSIS) was decreased in the islets of the Senp2-βKO mice without a significant change in insulin synthesis. Furthermore, islets of the Senp2-βKO mice exhibited enlarged mitochondria and lower oxygen consumption rates, accompanied by lower levels of S616 phosphorylated DRP1 (an active form of DRP1), a mitochondrial fission protein. Using a cell culture system of NIT-1, an islet β cell line, we found that increased SUMO2/3 conjugation to DRP1 due to SENP2 deficiency suppresses the phosphorylation of DRP1, which possibly induces mitochondrial dysfunction. In addition, SENP2 overexpression restored GSIS impairment induced by DRP1 knockdown and increased DRP1 phosphorylation. Furthermore, palmitate treatment decreased phosphorylated DRP1 and GSIS in β cells, which was rescued by SENP2 overexpression. These results suggest that SENP2 regulates mitochondrial function and insulin secretion at least in part by modulating the phosphorylation of DRP1 in pancreatic β cells.Subject terms: Sumoylation, Diabetes, Mechanisms of disease, Phosphorylation     

The disubstituted adamantyl derivative LW1564 inhibits the growth of cancer cells by targeting mitochondrial respiration and reducing hypoxia-inducible factor (HIF)-1α accumulation

Targeting cancer metabolism has emerged as an important cancer therapeutic strategy. Here, we describe the synthesis and biological evaluation of a novel class of hypoxia-inducible factor (HIF)-1α inhibitors, disubstituted adamantyl derivatives. One such compound, LW1564, significantly suppressed HIF-1α accumulation and inhibited the growth of various cancer cell lines, including HepG2, A549, and HCT116. Measurements of the oxygen consumption rate (OCR) and ATP production rate revealed that LW1564 suppressed mitochondrial respiration, thereby increasing the intracellular oxygen concentration to stimulate HIF-1α degradation. LW1564 also significantly decreased overall ATP levels by inhibiting mitochondrial electron transport chain (ETC) complex I and downregulated mammalian target of rapamycin (mTOR) signaling by increasing the AMP/ATP ratio, which increased AMP-activated protein kinase (AMPK) phosphorylation. Consequently, LW1564 promoted the phosphorylation of acetyl-CoA carboxylase, which inhibited lipid synthesis. In addition, LW1564 significantly inhibited tumor growth in a HepG2 mouse xenograft model. Taken together, the results indicate that LW1564 inhibits the growth of cancer cells by targeting mitochondrial ETC complex I and impairing cancer cell metabolism. We, therefore, suggest that LW1564 may be a potent therapeutic agent for a subset of cancers that rely on oxidative phosphorylation for ATP generation.Subject terms: Cancer metabolism, Cancer metabolism, Drug development     

Wafer-scale mitochondrial membrane potential assays

It has been reported that mitochondrial metabolic and biophysical parameters are associated with degenerative diseases and the aging process. To evaluate these biochemical parameters, current technology requires several hundred milligrams of isolated mitochondria for functional assays. Here, we demonstrate manufacturable wafer-scale mitochondrial functional assay lab-on-a-chip devices, which require mitochondrial protein quantities three orders of magnitude less than current assays, integrated onto 4' standard silicon wafer with new fabrication processes and materials. Membrane potential changes of isolated mitochondria from various well-established cell lines such as human HeLa cell line (Heb7A), human osteosarcoma cell line (143b) and mouse skeletal muscle tissue were investigated and compared. This second generation integrated lab-on-a-chip system developed here shows enhanced structural durability and reproducibility while increasing the sensitivity to changes in mitochondrial membrane potential by an order of magnitude as compared to first generation technologies. We envision this system to be a great candidate to substitute current mitochondrial assay systems.