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The current study intended to investigate the role of new natural compounds derived from the Sesuvium sesuvioides plant in mitigating symptoms of diabetes and insulin resistance in the diabetic mice model. Anti-advanced glycation activity, insulin, and adiponectin were quantified by enzyme-linked immunosorbent assay (ELISA). Glucose uptake was performed using enzymatic fluorescence assay, and glycogen synthesis was measured using PAS staining. Gene and protein expression was assessed using real time PCR (RT-PCR), and immunoblotting and fluorescent microscopy, respectively. The new flavonoid glycoside eupalitin 3- O -α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside 1 isolated from S. sesuvioides exhibited anti-AGE activity by reducing human glycated albumin in liver cells. In a diabetic mouse model treated with compound 1 , we observed improved glucose tolerance, increased adiponectin levels, and decreased insulin resistance. We also observed alleviated AGEs induced reduction in glucose uptake and restored glycogen synthesis in the compound 1 -treated diabetic mice muscles. Exploring the molecular mechanism of action in skeletal muscle tissue of diabetic mice, we found that 1 reduced AGE-induced reactive oxygen species and the inflammatory gene in the muscle of diabetic mice. Additionally, 1 exhibited these effects by reducing the gene and protein expression of receptor for advanced glycation end products (RAGE) and inhibiting protein kinase C (PKC) delta activation. This further led us to demonstrate that compound 1 reduced serine phosphorylation of IRS-1, thereby restoring insulin sensitivity. We conclude that a new flavonoid glycoside from S. sesuvioides could be a therapeutic target for the treatment of symptoms of insulin resistance and diabetes.

Objectives: Metabolic factors play significant role in the natural history of knee osteoarthritis (KO). There is a limited understanding of molecular and cellular events that give rise to the disease in patients. This study explored the possible cellular mechanisms by which metabolic syndrome leads to KO., Methods: This cross-sectional study enrolled 80 subjects with KO who fulfilled the ACR diagnostic criteria and were undergoing total knee replacement surgery. The patients were divided into two groups: KO patients without metabolic syndrome and KO patients with metabolic syndrome., Results: We hypothesised that metabolic syndrome may accelerate pathogenesis of OA by enhanced RAGE axis in articular cartilage and Infrapatellar fat pad of the knee joint. We have found enhanced protein expression of receptor for advanced glycation end products (RAGE) and its ligands AGEs and HMGB-1 in knee joint tissue of KO patients with metabolic syndrome as compared to KO patients without metabolic syndrome. Further downstream, the gene expression of oxidative stress regulators such as NADPH and inflammation, NFĸB were upregulated in KO patients with MetS as compared to KO patients alone. Higher levels of advanced oxidation products and inflammatory marker IL-17 were exhibited in synovial fluid of KO patients with metabolic syndrome. The enhanced levels of these oxidative stress and inflammatory markers were reflected in the serum of KO patients with metabolic syndrome as well., Conclusions: We conclude that enhanced function of RAGE axis could be one of the mechanisms by which metabolic syndrome leads to KO.

Objective: To compare the efficacies of common therapeutic regimens and their combinations, used in polycystic ovarian syndrome (PCOS) to improve fertility in reproductive-age women., Study Design: A descriptive study. Place and Duration of the Study: Department of Obstetric Gynaecologist, Medicare Cardiac and General Hospital, Karachi, Pakistan, from November 2022 to July 2023., Methodology: Out of 300 patients with the symptoms of menstrual irregularities and infertility, 152 were diagnosed as PCOS patients based on the ultrasound and hormonal assays and selected for study purpose. They were divided according to their therapeutic regimen into four treatment groups, treated by different therapeutic agents. Group A received metformin 500 mg/day (n = 38); Group B received metformin + myo-inositol 1g (n = 49); Group C received metformin + letrozole 2.5 mg (n = 36), and Group D received metformin + letrozole + myo-inositol (n = 29), orally for three months. All continuous variables, such as body mass index (BMI), FSH, LH, FT4, and FSI were analysed by applying t-test to all therapeutic groups, keeping p ≤0.05 as the level of significance., Results: HCG-positive was found as 86% (n = 33) in Group A, 63% (n = 31) in Group B, 52% (n = 19) in Group C, and 27% (n = 08) in Group D. There were statistically significant (p <0.001) changes in BMI, FSH, LH, FT4, and FSI as well. Metformin alone and metformin plus myo-inositol came out to be more effective than other regimens., Conclusion: Metformin alone and myo-inositol plus metformin are effective therapeutic options in PCOS-induced infertility problems., Key Words: Polycystic ovarian syndrome, Infertility, Metformin, Myo-inositol, Letrozole, Menstrual irregularities.

Background: There is a limited understanding of molecular and cellular events that derive disease progression in patients with corona virus disease 2019 (COVID-19). Receptor for advanced glycation end products (RAGE) is hyperactive in development and complications of several diseases by mediating oxidative stress and inflammation in the body. The present study aims to explore activation of RAGE signaling in patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with preexisting comorbidities, including hypertension and or diabetes. Methods: A total of 442 subjects with COVID-19, were recruited for the study. The molecular mechanism of Covid-19 was explored in blood cells, using ELISA, RT- PCR and Western blot. Results: Enhanced levels of ligands of RAGE, including AGEs, S100, and high-mobility group box-1 (HMGB-1) were observed in COVID-19 patients with severe diseases; however, their level was significantly higher in COVID-19 patients with comorbidities compared to COVID-19 patients without comorbidities. The expression of RAGE in parallel to ligands accumulation was significantly increased in patients with severe disease and comorbidities compared to COVID-19 patients with severe disease without comorbidities. The expression of downstream effectors of RAGE, including STAT-3 and nuclear factor kappa B (NF-kB), was also enhanced and their activity was increased in COVID-19 patients with comorbidities. Levels of inflammatory and oxidative stress biomarkers were markedly increased in COVID-19 patients with comorbidities. Conclusions: We conclude that upregulated RAGE axis plays critical role, to worsen the severity of the SARS-CoV-2 infection in patients with preexisting comorbidities and partly explain inflammatory and oxidative stress storm in severe COVID-19 patients.

Aims: Methylphenidate (MPD) widely prescribed for the treatment of attention deficit hyperactivity disorder (ADHD), is a psychostimulant and can produce addiction in patients treated with it. In view of growing increase in the use of drug by general population as a cognitive enhancer, the present study is designed to investigate reinforcing and cognition enhancing effects of MPD in rats. Associated changes in serotonin-1A receptor expression are investigated as a potential molecular mechanism involved., Methods: Learning acquisition and memory retention in Morris water-maze test were used to assess cognitive effects of MPD. Reinforcing effects were evaluated in conditioned place-preference (CPP) paradigm. The expression of 5-hydroxytryptamine (5-HT; serotonin)-1A receptor in the nucleus accumbens and prefrontal cortex of repeated MPD treated animals was determined by qRT-PCR., Findings: Lower doses (0.5 and 2.5 mg/kg) of MPD enhanced learning acquisition and memory retention but higher doses (5 mg/kg) impaired these. The drug administered repeatedly at a dose of 2.5 mg/kg was reinforcing in CPP test, but sensitization like effects of this dose were only transient. These animals tested in water-maze test exhibited improved memory retention but no effect occurred on learning acquisition. The expression of 5-HT-1A receptor was markedly attenuated in the nucleus accumbens; attenuation in the prefrontal cortex was not significant., Significance: The findings suggest that clinically relevant doses of MPD can produce drug addiction, but effects of the drug on memory retention are retained. A downregulation of 5-HT-1A receptor in the nucleus accumbens seems important in the reinforcing effects of MPD., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Accumulating evidence indicates that a number of tissues are damaged due to build-up of abnormal amount of Advanced Glycation End products (AGEs) in several diseases including diabetes. Currently AGE inhibitors are scarce in clinical use indicating a need for development of new anti-AGE agents. The aim of the current study is to identify the new AGE inhibitors and to decipher their mechanism of action for alleviating symptoms of diabetes in mice. Among several derivatives, one of the derivatives of indanedione, IDD-24 demonstrated highest inhibition of AGE formation and AGE mediated reactive oxygen species production in HepG-2 and mature 3T3-L1 adipocytes. In mice treated with IDD-24, reduction in serum AGE formation and expression of Receptor for AGEs (RAGE) was seen in IDD-24 treated db/db mice. In vivo, glycogen synthesis was also increased in muscle tissue. In adipocytes, anti-AGE agent restored AGEs' induced diminished glucose uptake in fat cells. Mice treated with IDD-24 exhibited increased glucose tolerance, increaed serum adiponectin levels and decreased insulin resistance. Deciphering mechanism of IDD-24 in diabetic mice, it was observed that nuclear factor-κB (NF-κB) and serine phosphorylation of Insulin receptor substrate-1 (IRS-1) declined, while diminished activation of c-Jun NH2-terminal kinase (JNK) appears to be partly responsible for restoration of insulin signaling. We conclude that IDD-24 can be a possible treatment target to address symptoms of diabetes., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Several members of cucurbitaceae family have been reported to regulate growth of cancer by interfering with STAT3 signaling. In the present study, we investigated the unique role and molecular mechanism of cucurbitacins (Cucs) in reducing symptoms of metabolic syndrome in mice. Cucurbitacin E (CuE) was found to reduce adipogenesis in murine adipocytes. CuE treatment diminished hypertrophy of adipocytes, visceral obesity and lipogenesis gene expression in diet induced mice model of metabolic syndrome (MetS). CuE also ameliorated adipose tissue dysfunction by reducing hyperleptinemia and TNF-alpha levels and enhancing hypoadiponectinemia. Results show that CuE mediated these effects by attenuating Jenus kinase- Signal transducer and activator of transcription 5 (JAK- STAT5) signaling in visceral fat tissue. As a result, CuE treatment also reduced PPAR gamma expression. Glucose uptake enhanced in adipocytes after stimulation with CuE and insulin resistance diminished in mice treated with CuE, as reflected by reduced glucose intolerance and glucose stimulated insulin secretion. CuE restored insulin sensitivity indirectly by inhibiting JAK phosphorylation and improving AMPK activity. Consequently, insulin signaling was up-regulated in mice muscle. As CuE positively regulated adipose tissue function and suppressed visceral obesity, dyslipedemia, hyperglycemia and insulin resistance in mice model of MetS, we suggest that CuE can be used as novel approach to treat metabolic diseases.

Diabetes is associated with neurodegeneration. Glycation ensues in diabetes and glycated proteins cause insulin resistance in brain resulting in amyloid plaques and NFTs. Also glycation enhances gliosis by promoting neuroinflammation. Currently there is no therapy available to target neurodegenration in brain therefore, development of new therapy that offers neuroprotection is critical. The objective of this study was to evaluate mechanistic effect of isatin derivative URM-II-81, an anti-glycation agent for improvement of insulin action in brain and inhibition of neurodegenration. Methylglyoxal induced stress was inhibited by treatment with URM-II-81. Also, Ser473 and Ser9 phosphorylation of Akt and GSK-3β respectively were restored by URM-II-81. Effect of URM-II-81 on axonal integrity was studied by differentiating Neuro2A using retinoic acid. URM-II-81 restored axonal length in MGO treated cells. Its effects were also studied in high fat and low dose streptozotocin induced diabetic mice where it reduced RBG levels and inhibited glycative stress by reducing HbA1c. URM-II-81 treatment also showed inhibition of gliosis in hippocampus. Histological analysis showed reduced NFTs in CA3 hippocampal region and restoration of insulin signaling in hippocampii of diabetic mice. Our findings suggest that URM-II-81 can be developed as a new therapeutic agent for treatment of neurodegenration., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Several reports indicate anti-hyperglycemic effects of Syzygium aromaticum. In the present study, we report for the first time that clove extract (SAM) and its compound nigricin (NGC) decreases free fatty acid-mediated insulin resistance in mouse myoblasts. In addition, NGC was able to diminish insulin resistance in a diabetic mouse model. We observed that SAM and its compound NGC exhibited significant antioxidant activity in murine skeletal muscle cells. They also modulated stress signaling by reducing p38 MAP kinase phosphorylation. NGC and SAM treatments enhanced proximal insulin signaling by decreasing serine phosphorylation of insulin receptor substrate-1 (IRS-1) and increasing its tyrosine phosphorylation. SAM and NGC treatments also modified distal insulin signaling by enhancing protein kinase B (PKB) and glycogen synthase kinase-3-beta (GSK-3 beta) phosphorylation in muscle cells. Glucose uptake was enhanced in muscle cells after treatment with SAM and NGC. We observed increased glucose tolerance, glucose-stimulated insulin secretion, decreased insulin resistance, and increased beta cell function in diabetic mice treated with NGC. The results of our study demonstrate that clove extract and its active agent NGC can be potential therapeutic agents for alleviating insulin resistance.

The present study aimed to decipher the mechanism of action of selected anti-diabetic plants extracts on palmitic acid mediated insulin resistance in muscle cells. Our results showed that extract from Peganum harmala seeds, Eucalyptus camaldulensis and Syzygium aromaticum leaves, showed significant antioxidant activity. We found that these extracts were able to affect stress signalling by reducing p-38 MAP kinase phosphorylation. They also reduced phosphorylation of substrate for insulin receptor (IRS) at serine residues and increased its phosphorylation at tyrosine residues and also enhanced PKB phosphorylation. Glucose uptake was also enhanced in muscle cells after treatment with these extracts. Extracts from Lantana camara, Psidium gujava fruit and different parts of Cassia alata did not affect FFA mediated down-regulation of insulin signalling. The study conclude that seeds of Peganum harmala and leaves of Eucalyptus camaldulensis and Syzygium aromaticum enhanced insulin signal transduction and glucose uptake in muscle cells via reducing oxidative stress. As a result, these herbal extracts may be considered useful to protect from insulin resistance.

Glycotoxins/Advanced glycation end products (AGEs) have implications in development of diabetes and related diseases. In the present study we deciphered the mechanisms of action of URM-II-81, a new derivative of isatin, in alleviation of insulin resistance in human hepatocytes and murine adipocytes. URM-II-81 reduced AGEs formation and receptor for advanced glycation end products (RAGE) expression in both cell types. We also observed suppression of methylglyoxal (MGO) mediated ROS production and deactivation of PKC-α. URM-II-81 restored proximal insulin signaling by modulating IRS-1 phosphorylation. URM-II-81 also alleviated MGO mediated diminished distal insulin signaling by increasing protein kinase B (PKB) and glycogen synthase kinase 3-beta (GSK-3-beta) phosphorylation. Glycogen synthesis was also increased in hepatocytes after treatment with URM-II-81. In adipocytes URM-II-81 prevented MGO induced reduced glucose uptake. We conclude that URM-II-81 can be a possible treatment target to address glycotoxins induced insulin resistance., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Methylglyoxal (MGO) is a highly reactive advanced glycation end products (AGEs) precursor and its abnormal accumulation causes damage to various tissues and organs. In our previous study, we synthesized a novel MGO inhibitor, MK-I-81, a bis-Schiff base derivative of isatin. In this study we demonstrate the mechanism of action of MK-I-81, on insulin resistance in skeletal muscle cells. MK-I-81 reduced AGEs formation and restored proximal insulin signaling by modulating IRS-1 phosphorylation. MK-I-81 also alleviated MGO mediated diminished distal insulin signaling by increasing protein kinase B and glycogen synthase kinase 3-beta phosphorylation. We also observed that MK-I-81 prevented reduced glucose uptake and glycogen synthesis induced by MGO in muscle cells. We found that the mechanism of action by which MK-I-81 reduced insulin resistance was suppression of production of MGO mediated ROS production in C2C12 cells. We evaluated deactivation of PKC-α and receptor for advanced glycation end products (RAGE) after treatment of cells with MK-I-81. MK-I-81 also reduced MGO mediated IRS-1, PKC-α and RAGE interaction in muscle cells. MK-I-81 also promoted nuclear factor erythroid 2-related factor-2 phosphorylation, heme oxygenase-1 and glyoxalase expression levels. We conclude that MK-I-81 can be a potential therapeutic target to address AGEs mediated insulin resistance. A novel Advanced Glycation End products (AGEs) inhibitor, MK-I-81 (a bis Schiff base of isatin), restored AGEs mediated down regulation of insulin signaling via modulating key molecules of proximal and distal insulin signaling. MK-I-81 also increased glucose uptake and glycogen synthesis in muscle cells. Novel bis-Schiff base of isatin showed significant antioxidant activity and also reduced receptor for AGEs (RAGE) expression and PKC-alpha activation therefore; MK-I-81 reduces AGEs induced insulin resistance.

In mammals, the male and female hormones androgen and estrogen act as endocrine regulators of energy metabolism. However, adipose tissue is also a site of androgen and estrogen synthesis; androgens convert to estrogens in these tissues, and adipose tissue is also a reservoir of steroids that act locally in a paracrine and intracrine manner. Thus, in adipose tissue, the local output of sex hormones is more complex than would be suggested by routine measurement of serum hormone concentrations. This review integrates studies on the effects of androgens and estrogens in the developmental programming of adipose tissue function in early life and addresses the contributions of local androgen and estrogen metabolism on adipose tissue function in adults.

Among women, the polycystic ovarian syndrome (PCOS) is considered a form of metabolic syndrome with reproductive abnormalities. Women with PCOS show increased sympathetic tone, visceral adiposity with enlarged adipocytes, hypoadiponectinemia, insulin resistance, glucose intolerance, increased inactive osteocalcin, and hypertension. Excess fetal exposure to androgens has been hypothesized to play a role in the pathogenesis of PCOS. Previously, we showed that neonatal exposure to the androgen testosterone (NT) programs leptin resistance in adult female mice. Here, we studied the impact of NT on lean and adipose tissues, sympathetic tone in cardiometabolic tissues, and the development of metabolic dysfunction in mice. Neonatally androgenized adult female mice (NTF) displayed masculinization of lean tissues with increased cardiac and skeletal muscle as well as kidney masses. NTF mice showed increased and dysfunctional white adipose tissue with increased sympathetic tone in both visceral and subcutaneous fat as well as increased number of enlarged and insulin-resistant adipocytes that displayed altered expression of developmental genes and hypoadiponectinemia. NTF exhibited dysfunctional brown adipose tissue with increased mass and decreased energy expenditure. They also displayed decreased undercarboxylated and active osteocalcin and were predisposed to obesity during chronic androgen excess. NTF showed increased renal sympathetic tone associated with increased blood pressure, and they developed glucose intolerance and insulin resistance. Thus, developmental exposure to testosterone in female mice programs features of cardiometabolic dysfunction, as can be observed in women with PCOS, including increased sympathetic tone, visceral adiposity, insulin resistance, prediabetes, and hypertension.

In mammals, males consume more food, which is considered a masculinized behavior, but the underlying mechanism of this sex-specific feeding behavior is unknown. In mice, neonatal testosterone (NT) is critical to masculinize the developing brain, leading to sex differences in reproductive physiology. The proopiomelanocortin (POMC) neurons of the hypothalamic arcuate nucleus (ARC) are critical to suppress energy intake and POMC innervation of hypothalamic feeding circuits develops to a large extent neonatally. We hypothesized that NT programs the masculinization of energy intake by programming POMC neurons. We tested this hypothesis by comparing control females and control males (CMs) with female mice neonatally androgenized with testosterone (NTFs). We show that increased food intake in CMs is associated with reduced POMC expression and decreased intensity of neuronal projections from POMC neurons within the ARC compared with control females. We found that NTFs display a masculinized energy intake and ARC POMC expression and innervation as observed in CMs, which can be mimicked by neonatal exposure to the androgen receptor agonist dihydrotestosterone (DHT). NTFs also exhibit hyperleptinemia and a decreased ability of leptin to up-regulate POMC, suppress food intake, and prevent adipose tissue accumulation, independent of signal transducer and activator of transcription 3. However, this leptin resistance is specific to NTFs, is not a consequence of masculinization, and is reproduced by neonatal exposure to estrogen but not DHT. Thus, NT programs a sexual differentiation of POMC neurons in female mice via DHT but also predisposes to leptin resistance and obesity in an estrogen-dependent manner.

Antibodies that recognize specifically phosphorylated sites on proteins are widely utilized for studying the regulation and biological function of phosphoproteins. The proposed strategy is a powerful, analytical tool allowing the generation of phospho-site specific antibodies albeit adjacent phosphorylation sites are present. Here, we demonstrate the assessment and elimination of cross reactivity of phospho-site-specific-Ser(357) IRS-1 antibody. While determining the specificity of p-Ser(357) antiserum we came across the cross reactivity of the antiserum with adjacent Ser(358) which was successfully abolished by an improved immuno-purification method. The specificity of the purified antiserum was then verified by indirect ELISA, results of ELISA were also mirrored in the experiments carried out in BHK-IR cells using different mutants of IRS-1 carrying mutations at either Ser(357)/Ser(358)/Ser(357/358). Immuno-purified-p-Ser(357) did not react with IRS-1 Ala(357) and IRS-1 Ala(357/358). In conclusion, the present study describes generation and characterization of p-Ser(357) IRS-1 antibody, which reacts with IRS-1 in site specific and phosphorylation state-dependent manner without showing cross reactivity to adjacent Ser(358). This antibody can be effectively used to further clarify the inhibitory role of Ser(357) in insulin signal transduction.

The activation of the protein kinase C (PKC) family of serine/threonine kinases contributes to the modulation of insulin signaling, and the PKC-dependent phosphorylation of insulin receptor substrate (IRS)-1 has been implicated in the development of insulin resistance. Here we demonstrate Ser(357) of rat IRS-1 as a novel PKC-delta-dependent phosphorylation site in skeletal muscle cells upon stimulation with insulin and phorbol ester using Ser(P)(357) antibodies and active and kinase dead mutants of PKC-delta. Phosphorylation of this site was simulated using IRS-1 Glu(357) and shown to reduce insulin-induced tyrosine phosphorylation of IRS-1, to decrease activation of Akt, and to subsequently diminish phosphorylation of glycogen synthase kinase-3. When the phosphorylation was prevented by mutation of Ser(357) to alanine, these effects of insulin were enhanced. When the adjacent Ser(358), present in mouse and rat IRS-1, was mutated to alanine, which is homologous to the human sequence, the insulin-induced phosphorylation of glycogen synthase kinase-3 or tyrosine phosphorylation of IRS-1 was not increased. Moreover, both active PKC-delta and phosphorylation of Ser(357) were shown to be necessary for the attenuation of insulin-stimulated Akt phosphorylation. The phosphorylation of Ser(357) could lead to increased association of PKC-delta to IRS-1 upon insulin stimulation, which was demonstrated with IRS-1 Glu(357). Together, these data suggest that phosphorylation of Ser(357) mediates at least in part the adverse effects of PKC-delta activation on insulin action.