The effects of nitric oxide on soybean superoxide dismutase activity during osmotic stress

>Magister Scientiae - MScNitric oxide (NO) is a signaling molecule involved in mediating plant responses to various biotic and abiotic stresses. Major abiotic stresses (drought, salinity, cold) induce common cellular responses, causing osmotic stress in plants. This results in oxidative stress due to increased production of reactive oxygen species (ROS). The increased ROS levels simultaneously induce the antioxidative system (including antioxidant enzymes such as superoxide dismutase) that regulates ROS toxicity and enhance stress tolerance in plants. It is suggested that the scavenging of ROS by antioxidant enzymes can be controlled by NO. The aim of this study was to evaluate the role of exogenously applied NO on soybean (Glycine max L. Merr.) during osmotic stress, with the purpose of determining the effects of NO on the superoxide dismutase (SOD) activity in response to osmotic stress. This study also aimed at identifying and characterising SOD isoforms induced in soybean in response to osmotic stress and exogenous NO. To achieve these aims, soybean plants were treated with sorbitol (to induce osmotic stress), an NO donor [2,2'-(hydroxynitrosohydrazono)bis-ethanimine, DETA/NO] and its respective control (Diethylenetriamine, DETA). The results showed that exogenous NO alleviated osmotic stress-induced damage by reducing the superoxide radical content, lipid peroxidation levels and also maintaining cell viability in soybean leaves, nodules and roots. Only two SOD isoforms i.e. manganese SOD (MnSOD) and copper/zinc SOD (CuZnSOD) were identified and characterised in soybean leaves and roots, iron SOD (FeSOD) was not induced. The isoforms identified exhibited low SOD activity in response to osmotic stress, with the exception of a few isoforms that had increased activity. The SOD activity was regulated by exogenously applied NO. The enzymatic activity of SOD isoforms was up-regulated by exogenous NO, except for a few SOD isoforms that were not responsive to NO. The results also showed that the increased SOD activity was associated with reduced lipid peroxidation levels. The results obtained from this study suggest that exogenous NO improves osmotic stress tolerance in soybean by regulating and increasing the SOD activity of only specific isoforms. The increased SOD activity maintains the redox homeostasis balance by detoxifying and controlling the superoxide radical levels, subsequently reducing lipid peroxidation and maintaining cell viability

Changes in subcutaneous adipose tissue microRNA expression in response to exercise training in African women with obesity

The mechanisms that underlie exercise-induced adaptations in adipose tissue have not been elucidated, yet, accumulating studies suggest an important role for microRNAs (miRNAs). This study aimed to investigate miRNA expression in gluteal subcutaneous adipose tissue (GSAT) in response to a 12-week exercise intervention in South African women with obesity, and to assess depot-specific differences in miRNA expression in GSAT and abdominal subcutaneous adipose tissue (ASAT). In addition, the association between exercise-induced changes in miRNA expression and metabolic risk was evaluated. Women underwent 12-weeks of supervised aerobic and resistance training (n = 19) or maintained their regular physical activity during this period (n = 12). Exercise-induced miRNAs were identified in GSAT using Illumina sequencing, followed by analysis of differentially expressed miRNAs in GSAT and ASAT using quantitative real-time PCR. Associations between the changes (pre- and postexercise training) in miRNA expression and metabolic parameters were evaluated using Spearman’s correlation tests. Exercise training significantly increased the expression of miR-155-5p (1.5-fold, p = 0.045), miR-329-3p (2.1-fold, p < 0.001) and miR-377-3p (1.7-fold, p = 0.013) in GSAT, but not in ASAT. In addition, a novel miRNA, MYN0617, was identified in GSAT, with low expression in ASAT. The exercise-induced differences in miRNA expression were correlated with each other and associated with changes in high-density lipoprotein concentrations. Exercise training induced adipose-depot specific miRNA expression within subcutaneous adipose tissue depots from South African women with obesity. The significance of the association between exercise-induced miRNAs and metabolic risk warrants further investigation.The South African Medical Research Council (SAMRC) and the National Research Foundation of South Africa (NRF), Competitive Programme for Rated Researchers.http://www.nature.com/scientificreportsam2023Obstetrics and Gynaecolog

An investigation into the anti-obesity properties of Cyclopia

Thesis (PhD)--Stellenbosch University, 2016.ENGLISH SUMMARY: Polyphenols have a range of health promoting effects against chronic diseases such as obesity and type 2 diabetes mellitus. Previous research in our group showed that Cyclopia species (honeybush), endemic South African plants, display anti-obesity effects. The aim of this study was to investigate the anti-obesity potential of polyphenol-enriched extracts of three Cyclopia spp. (C. subternata, C. intermedia and C. maculata) using bioactivity guided fractionation to facilitate the identification of anti-obesity polyphenols. Aqueous methanol extracts of C. subternata, C. intermedia and C. maculata were prepared and separated into their aqueous and organic fractions, which were tested for their anti-obesity effects in 3T3-L1 adipocytes using the Oil Red O, glycerol release (marker of lipolysis), and triglyceride assays. Cytotoxicity was evaluated using the 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and adenosine triphosphate (ATP) assays. The anti-obesity properties of the organic fraction of the aqueous methanol extract of C. intermedia were evaluated in obese Leprdb/db mice. Thereafter, the organic fraction was separated into four fractions (CCC F1 to CCC F4) using high performance counter-current chromatography (HPCCC), which were tested for their anti-obesity and cytotoxic effects in 3T3-L1 pre-adipocytes and in mature adipocytes. Liquid chromatography tandem mass spectrometry (LC-MS/MS) and quantitative high performance liquid chromatography diode array detection (HPLC-DAD) were used for determination of phenolic composition. The molecular mechanisms of action of the organic fraction and its CCC fractions were assessed with quantitative real-time PCR and western blot analysis. The polyphenol-enriched extracts of C. subternata, C. intermedia and C. maculata, and their aqueous and organic fractions exhibited differences in phenolic composition and bioactivity. The aqueous fractions of C. maculata and C. subternata, containing lower phenolic content than the organic fractions, decreased lipid content in 3T3-L1 adipocytes compared to their organic counterparts. However, the organic fraction of C. intermedia, with higher phenolic content than its aqueous fraction, decreased lipid content compared to its aqueous fraction in 3T3-L1 adipocytes. In Leprdb/db mice, the organic fraction of C. intermedia decreased body weight gain, without affecting food or water consumption. Further fractionation of the organic fraction of C. intermedia by HPCCC provided four fractions with differences in phenolic composition and anti-obesity effects. None of the extracts or fractions, except for the aqueous C. intermedia and organic C. maculata fractions which decreased ATP content, affected cell viability as measured with the MTT and ATP assays during acute treatment. The highest concentrations of CCC F1 and CCC F3 decreased ATP content during chronic treatment in pre-adipocytes. In 3T3-L1 adipocytes, the organic fraction of C. intermedia and CCC fractions modulated the expression of genes implicated in lipid and energy metabolism, whereas the organic fraction of C. intermedia had no effect on the expression of these genes in Leprdb/db mice. In conclusion, the organic fraction of C. intermedia exhibited anti-obesity properties in vitro and in vivo. A flavanone with anti-obesity potential, neoponcirin, was identified in C. intermedia, the first discovery of this compound in Cyclopia spp. Bioactivity guided fractionation of the organic fraction of C. intermedia resulted in CCC fractions retaining different polyphenols with varying anti-obesity effects. These results demonstrate that the anti-obesity potential of C. intermedia is due to the presence of more than one compound, with different mechanisms of action, or the synergistic effects of more than one compound, may contribute to the bioactivity of C. intermedia.AFRIKAANSE OPSOMMING: Polifenole het ‘n verskeidenheid van voordelige gesondheids-effekte teen chroniese siekte toestande wat obesiteit en diabetes mellitus tipe 2 insluit. Voorafgaande navorsing in ons groep het aangedui dat die uitsluitlik inheems Suid-Afrikaanse Cyclopia-spesies (heuningbos), obesiteits-teenwerkende effekte toon. Die mikpunt van die huidige studie was om die obesiteits-teenwerkende potensiaal van polifenol-verrykte ekstrakte van drie Cyclopia-spesies (C. subternata, C. intermedia en C. maculata) te ondersoek deur bio-aktiwiteitsgerigte fraksionering te gebruik om obesiteits-teenwerkende polifenole te identifiseer. Waterige metanol ekstrakte van C. subternata, C. intermedia en C. maculata was voorberei en geskei in water-oplosbare en organiese fraksies wat dan getoets was vir moontlike obesiteits-teenwerkende eienskappe in 3T3-L1 vetselle deur gebruik te maak van “Oil Red O”, gliserol-vrystelling (‘n merker vir lipolise) en trigliseried toetse. Seldodende effekte is ondersoek deur die 3-[4,5-Di-metielthiazool-2-iel]-2,5-difenieltetrazolium bromied (MTT) and adenosien trifosfaat (ATP) toetse te gebruik. Die obesiteits-teenwerkende effekte van die organiese fraksie verkry vanuit die waterige metanol ekstrak van C. intermedia is verder ondersoek in Leprdb/db-muise. Hierna is die organiese fraksie opgedeel in vier fraksies (CCC F1 tot CCC F4) deur hoë-werkverrigting teen-vloei chromatografie (HPCCC) te gebruik en dan weer getoets vir obesiteits-teenwerkende en seldodende effekte in 3T3-L1 pre-vetselle en volwasse vetselle. Vloeistof-chromatografie gekoppelde massaspektrometrie (LC-MS/MS) en kwantitatiewe hoë-werkverrigting vloeistof-chromatografie met diode-reeks deteksie (HPLC-DAD) is gebruik vir die bepaling van die fenoliese samestelling. Die molekulêre werkingsmeganismes van die organiese fraksie en die CCC-fraksies is ondersoek deur die kwantitatiewe “intyds” polimerase kettingreaksie en western-klad tegnieke te gebruik. Die polifenol-verrykte ekstrakte van C. subternata, C. intermedia en C. maculata en hul wateroplosbare en organiese fraksies het verskille getoon in fenoliese samestelling asook bio-aktiwiteit. Die wateroplosbare fraksies van C. maculata en C. subternata, met laer fenoliese inhoud as die organiese fraksies, het vet-inhoud in 3T3-L1 vetselle verlaag relatief tot hul ooreenstemmende organiese fraksies. Die organiese fraksie van C. intermedia, met hoër fenoliese inhoud as die wateroplosbare fraksie, het egter die vetinhoud van die 3T3-L1 vetselle verlaag in vergelyking met die ooreenstemmende wateroplosbare fraksie. In Leprdb/db-muise het die organiese fraksie van C. intermedia ‘n verlaging in liggaamsgewigstoename tot gevolg gehad sonder om die voedsel- of water-inname te beïnvloed. Verdere skeiding van die organiese fraksie van C. intermedia met HPCCC het 4 fraksies opgelewer wat elkeen verskil het van die ander in terme van fenoliese samestelling en obesiteits-teenwerkende effekte. Geen van die ekstrakte of fraksies, behalwe die wateroplosbare fraksie van C. intermedia en die organiese fraksie van C. maculata wat ATP-inhoud laat daal het in 3T3-L1 vetselle, het sel-lewensvatbaarheid beïnvloed soos gemeet in MTT- en ATP-toetse na akute behandeling nie. Tydens chroniese behandeling van 3T3-L1 pre-vetselle, het die hoogste konsentrasies van CCC F1 en CCC F3 die ATP-inhoud verlaag. In 3T3-L1 vetselle, het die organiese fraksie van C. intermedia en daaropvolgende CCC-fraksies die uitdrukking van gene betrokke by vet en energie metabolisme gemoduleer terwyl die organiese fraksie van C. intermedia nie die uitdrukking van hierdie gene in Leprdb/db-muise beïnvloed het nie. In samevatting, die organiese fraksie van C. intermedia het obesiteits-teenwerkende eienskappe getoon in vitro en in vivo. ‘n Flavanoon met obesiteits-teenwerkende potensiaal, neo-ponsirien, is geidentifiseer in C. intermedia en is vir die eerste keer gevind in Cyclopia spesies. Bio-aktiwiteitsgerigte fraksionering van die organiese fraksie van C. intermedia het CCC-fraksies gelewer wat verskillende polifenole bevat het en wat verskillende obesiteits-teenwerkende effekte getoon het. Hierdie resultate dui daarop dat die obesiteits-teenwerkende potensiaal van C. intermedia nie toegeken kan word aan ‘n enkele fenoliese verbinding nie, maar dat sinergistiese of kumulatiewe effekte vanaf meer as een obesiteits-teenwerkende verbinding, met verskille in werkingsmeganisme, betrokke mag wees

Adiponectin as a potential biomarker for pregnancy disorders

Adiponectin is an adipocyte-derived hormone that plays a critical role in energy homeostasis, mainly attributed to its insulin-sensitizing properties. Accumulating studies have reported that adiponectin concentrations are decreased during metabolic diseases, such as obesity and type 2 diabetes, with an emerging body of evidence providing support for its use as a biomarker for pregnancy complications. The identification of maternal factors that could predict the outcome of compromised pregnancies could act as valuable tools that allow the early recognition of high-risk pregnancies, facilitating close follow-up and prevention of pregnancy complications in mother and child. In this review we consider the role of adiponectin as a potential biomarker of disorders associated with pregnancy. We discuss common disorders associated with pregnancy (gestational diabetes mellitus, preeclampsia, preterm birth and abnormal intrauterine growth) and highlight studies that have investigated the potential of adiponectin to serve as biomarkers for these disorders. We conclude the review by recommending strategies to consider for future research.This research was funded by South African Medical Research Council.The South African Medical Research Councilhttps://www.mdpi.com/journal/ijmsam2022Obstetrics and Gynaecolog

Time- and glucose-dependent differentiation of 3T3-L1 adipocytes mimics dysfunctional adiposity

The 3T3-L1 murine adipocyte cell line remains one of the most widely used models to study the mechanisms of obesity and related pathologies. Most studies investigate such mechanisms using mature adipocytes that have been chemically induced to differentiate for 7 days in media containing 25 mM glucose. However, the dysfunctional characteristics commonly observed in obesity including adipocyte hypertrophy, increased expression of inflammatory markers, enhanced production of reactive oxygen species (ROS), increased steroidogenic enzyme expression/activity and production of steroid hormones, are not necessarily mimicked in these cells. The aim of this study was to provide an inexpensive model which represents the well-known characteristics of obesity by manipulating the time of adipocyte differentiation and increasing the concentration of glucose in the cell media. Our results showed a glucoseand time-dependent increase in adipocyte hypertrophy, ROS production and gene expression of the proinflammatory cytokine interleukin-6 (IL-6), as well as a time-dependent increase in lipolysis and in the gene expression of the chemokine monocyte chemoattractant protein 1 (MCP1). We also showed that gene expression of the steroidogenic enzymes 11-beta-hydroxysteroid dehydrogenase type 1 (11bHSD1), 17bHSD type 7 and 12, as well as CYP19A1 (aromatase), were significantly higher in the hypertrophic model relative to the control adipocytes differentiated using the conventional method. The increase in 11bHSD1 and 17bHSD12 expression was consistent with the enhanced conversion of cortisone and androstenedione to cortisol and testosterone, respectively. As these characteristics reflect those commonly observed in obesity, hypertrophic 3T3-L1 adipocytes are an appropriate in vitro model to study mechanisms of adipocyte dysfunction in an era where the rise in obesity incidence is a global health concern, and where access to adipose tissue from obese patients are limited.http://www.elsevier.com/locate/ybbrcObstetrics and Gynaecolog

A dose-dependent effect of dimethyl sulfoxide on lipid content, cell viability and oxidative stress in 3T3-L1 adipocytes

Dimethyl sulfoxide (DMSO) is an effective solvent and cytoprotectant agent that can induce diverse actions in experimental settings, ranging from metabolic stress to cytotoxic effects depending on the concentration used. Therefore, for the quality of experiments and reproducibility of results it is essential to establish a precise and non-toxic dose of DMSO within a specific cell system. 3T3-L1 adipocytes, represent a well-established in vitro cell model used to assess the anti-obesity potential of extracts and compounds. Although DMSO is commonly used as a solvent for these experiments, there is limited data available on the compounding effects of using DMSO. The purpose of this study was to assess a concentration-dependent effect of DMSO on lipid content, cell viability and oxidative damage in 3T3-L1 adipocytes. Results showed that DMSO at doses ≥ 0.1% increased mitochondrial membrane potential as measured by JC-1 fluorescent staining, while doses ≥ 10% reduced the lipid content in matured adipocytes. Consistently, higher doses significantly reduced cell viability, elevated reactive oxygen species levels, depleted intracellular glutathione levels, and accelerated apoptosis and cell necrosis. An interesting finding was that a DMSO dose of 0.01% improved glutathione content of 3T3-L1 adipocytes and had minimal effects on cell viability, apoptosis or and necrosis, supporting its antioxidant effect. Therefore, this study provides compelling evidence that precaution should be taken when assessing compounds dissolved in DMSO, particularly doses ≥1% that were shown to induce oxidative stress in 3T3-L1 adipocytes. Keywords: Dimethyl sulfoxide, Cell viability, Oxidative stress, Apoptosis, 3T3-L1 adipocyte

Therapeutic Effects of Curcumin Derivatives against Obesity and Associated Metabolic Complications: A Review of In Vitro and In Vivo Studies

Obesity is a major cause of morbidity and mortality globally, increasing the risk for chronic diseases. Thus, the need to identify more effective anti-obesity agents has spurred significant interest in the health-promoting properties of natural compounds. Of these, curcumin, the most abundant and bioactive constituent of turmeric, possesses a variety of health benefits including anti-obesity effects. However, despite its anti-obesity potential, curcumin has demonstrated poor bioavailability, which limits its clinical applicability. Synthesizing curcumin derivatives, which are structurally modified analogs of curcumin, has been postulated to improve bioavailability while maintaining therapeutic efficacy. This review summarizes in vitro and in vivo studies that assessed the effects of curcumin derivatives against obesity and its associated metabolic complications. We identified eight synthetic curcumin derivatives that were shown to ameliorate obesity and metabolic dysfunction in diet-induced obese animal models, while five of these derivatives also attenuated obesity and associated metabolic complications in cell culture models. These curcumin derivatives modulated adipogenesis, lipid metabolism, insulin resistance, steatosis, lipotoxicity, inflammation, oxidative stress, endoplasmic reticulum stress, apoptosis, autophagy, fibrosis, and dyslipidemia to a greater extent than curcumin. In conclusion, the findings from this review show that compared to curcumin, synthetic curcumin derivatives present potential candidates for further development as therapeutic agents to modulate obesity and obesity-associated metabolic complications

Adipose tissue as a possible therapeutic target for polyphenols : a case for Cyclopia extracts as anti-obesity nutraceuticals

CITATION: Jack, B. U., et al. 2019. Adipose tissue as a possible therapeutic target for polyphenols : a case for Cyclopia extracts as anti-obesity nutraceuticals. Biomedicine and Pharmacotherapy, 120:109439, doi:1016/j.biopha.2019.109439.The original publication is available at https://www.sciencedirect.comENGLISH ABSTRACT: Obesity is a significant contributor to increased morbidity and premature mortality due to increasing the risk of many chronic metabolic diseases such as type 2 diabetes, cardiovascular disease and certain types of cancer. Lifestyle modifications such as energy restriction and increased physical activity are highly effective first-line treatment strategies used in the management of obesity. However, adherence to these behavioral changes is poor, with an increased reliance on synthetic drugs, which unfortunately are plagued by adverse effects. The identification of new and safer anti-obesity agents is thus of significant interest. In recent years, plants and their phenolic constituents have attracted increased attention due to their health-promoting properties. Amongst these, Cyclopia, an endemic South African plant commonly consumed as a herbal tea (honeybush), has been shown to possess modulating properties against oxidative stress, hyperglycemia, and obesity. Likewise, several studies have reported that some of the major phenolic compounds present in Cyclopia spp. exhibit anti-obesity effects, particularly by targeting adipose tissue. These phenolic compounds belong to the xanthone, flavonoid and benzophenone classes. The aim of this review is to assess the potential of Cyclopia extracts as an anti-obesity nutraceutical as underpinned by in vitro and in vivo studies and the underlying cellular mechanisms and biological pathways regulated by their phenolic compounds.https://www.sciencedirect.com/science/article/pii/S0753332219330562Publisher's versio

Evaluating the Therapeutic Potential of Curcumin and Synthetic Derivatives: A Computational Approach to Anti-Obesity Treatments

Natural compounds such as curcumin, a polyphenolic compound derived from the rhizome of turmeric, have gathered remarkable scientific interest due to their diverse metabolic benefits including anti-obesity potential. However, curcumin faces challenges stemming from its unfavorable pharmacokinetic profile. To address this issue, synthetic curcumin derivatives aimed at enhancing the biological efficacy of curcumin have previously been developed. In silico modelling techniques have gained significant recognition in screening synthetic compounds as drug candidates. Therefore, the primary objective of this study was to assess the pharmacokinetic and pharmacodynamic characteristics of three synthetic derivatives of curcumin. This evaluation was conducted in comparison to curcumin, with a specific emphasis on examining their impact on adipogenesis, inflammation, and lipid metabolism as potential therapeutic targets of obesity mechanisms. In this study, predictive toxicity screening confirmed the safety of curcumin, with the curcumin derivatives demonstrating a safe profile based on their LD50 values. The synthetic curcumin derivative 1A8 exhibited inactivity across all selected toxicity endpoints. Furthermore, these compounds were deemed viable candidate drugs as they adhered to Lipinski’s rules and exhibited favorable metabolic profiles. Molecular docking studies revealed that both curcumin and its synthetic derivatives exhibited favorable binding scores, whilst molecular dynamic simulations showed stable binding with peroxisome proliferator-activated receptor gamma (PPARγ), csyclooxygenase-2 (COX2), and fatty acid synthase (FAS) proteins. The binding free energy calculations indicated that curcumin displayed potential as a strong regulator of PPARγ (−60.2 ± 0.4 kcal/mol) and FAS (−37.9 ± 0.3 kcal/mol), whereas 1A8 demonstrated robust binding affinity with COX2 (−64.9 ± 0.2 kcal/mol). In conclusion, the results from this study suggest that the three synthetic curcumin derivatives have similar molecular interactions to curcumin with selected biological targets. However, in vitro and in vivo experimental studies are recommended to validate these findings

Inflammation and Oxidative Stress in an Obese State and the Protective Effects of Gallic Acid

Metabolic complications in an obese state can be aggravated by an abnormal inflammatory response and enhanced production of reactive oxygen species. Pro-inflammatory response is known to be associated with the formation of toxic reactive oxygen species and subsequent generation of oxidative stress. Indeed, adipocytes from obese individuals display an altered adipokine profile, with upregulated expression and secretion of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-&alpha;) and interleukin (IL-6). Interestingly, natural compounds, including phenolic enriched foods are increasingly explored for their ameliorative effects against various metabolic diseases. Of interest is gallic acid, a trihydroxybenzoic acid that has progressively demonstrated robust anti-obesity capabilities in various experimental models. In addition to reducing excessive lipid storage in obese subjects, gallic acid has been shown to specifically target the adipose tissue to suppress lipogenesis, improve insulin signaling, and concomitantly combat raised pro-inflammatory response and oxidative stress. This review will revise mechanisms involved in the pathophysiological effects of inflammation and oxidative stress in an obese state. To better inform on its therapeutic potential and improvement of human health, available evidence reporting on the anti-obesity properties of gallic acid and its derivatives will be discussed, with emphases on its modulatory effect on molecular mechanisms involved in insulin signaling, inflammation and oxidative stress