Altered Igf2 imprint leads to accelerated adipogenesis and early onset of metabolic syndrome in male mice following gestational arsenic exposure.

Developmental exposure to environmental pollutants has been shown to promote adverse health outcomes in offspring. Exposure to heavy metals such as arsenic which also has endocrine-disrupting activity is being increasingly linked with cancers, diabetes, and lately with Metabolic Syndrome (MetS). In this work, we have assessed the effects of preconceptional plus gestational arsenic exposure on the developmental programming of MetS in offspring. In our study, only gestational arsenic exposure led to reduced birth weight, followed by catch-up growth, adiposity, elevated serum triglycerides levels, and hyperglycemia in male offspring. Significant adipocyte dysfunction was observed in offspring with increased hypertrophy, insulin resistance, and chronic inflammation in epididymal white adipose tissue. Adipose tissue regulates the metabolic health of individuals and its dysfunction resulted in elevated serum levels of metabolism-regulating adipokines (Leptin, Resistin) and pro-inflammatory cytokines (PAI-1, TNFα). The progenitor adipose-derived stem cells (AdSCs) from exposed progeny had increased proliferation and adipogenic potential with excess lipid accumulation. We also found increased activation of Akt, ERK1/2 & p38 MAPK molecules in arsenic-exposed AdSCs along with increased levels of phospho-Insulin-like growth factor-1 receptor (p-IGF1R) and its upstream activator Insulin-like growth factor-2 (IGF2). Overexpression of Igf2 was found to be due to arsenic-mediated DNA hypermethylation at the imprinting control region (ICR) located -2kb to -4.4 kb upstream of the H19 gene which caused a reduction in the conserved zinc finger protein (CTCF) occupancy. This further led to persistent activation of the MAPK signaling cascade and enhanced adipogenesis leading to the early onset of MetS in the offspring.

Anti-adipogenic action of a novel oxazole derivative through activation of AMPK pathway.

Obesity is a chronic disorder with multifactorial etiology, including genetic, medical, dietary and other environmental factors. Both natural and synthetic heterocyclic compounds, especially oxazoles, represent an interesting group of compounds and have gained much attention due to their remarkable biological activities. Therefore, a library of 3,3-DMAH (3,3-dimethylallylhalfordinol) inspired N-alkylated oxazole bromide salts with varied substitutions were prepared and screened using the 3T3-L1 model of adipogenesis and HFD-induced obesity model in Syrian golden hamsters. Several compounds in the synthesized series displayed remarkable anti-adipogenic potential on the differentiation of 3T3-L1 preadipocytes. Compound 19e, displayed the most potent activity of all and selected for further studies. Compound 19e inhibited mitotic clonal expansion of 3T3-L1 cells and enhanced the mitochondrial oxygen consumption rate of the cells during early phase of differentiation via AMPK activation. 19e also improved the dyslipidaemia in high calorie diet fed Syrian Golden Hamsters. Therefore, compound 19e can serve as a potential lead against adipogenesis and dyslipidaemia models and could be further investigated to affirm its significance as a drug candidate.

16-Hydroxy-ent-halima-5(10),13-dien-15,16-olide from Polyalthia longifolia targets adipogenesis by inhibiting mitotic clonal expansion and ameliorates dyslipidemia.

Obesity-related metabolic disorders are increasing at an alarming rate worldwide. The FDA has approved many molecules for weight loss therapy; most of them act on the gut level by inhibiting lipid uptake or on the central nervous system by controlling appetite. Limitations and drawbacks have propelled the search for new pharmacophores exhibiting favourable metabolic alteration at adipocytes, and natural products have always been there to prove their worth. In our efforts, we have identified 16-hydroxy-ent-halima-5(10),13-dien-15,16-olide (PLH), a halimane diterpene isolated from Polyalthia longifolia, demonstrating anti-adipogenic and anti-dyslipidemic activity. It inhibited adipogenesis in 3T3-L1 preadipocyte and C3H10T1/2 mesenchymal stem cell lines. Furthermore, it decreased set of adipogenic markers at transcript and protein levels. Cell cycle studies indicated that PLH halts the mitotic clonal expansion. Mechanistic studies shows that PLH activate Wnt/ß-catenin signaling pathway to inhibit the adipogenesis. The study suggested that PLH inhibited adipogenesis during the early phase of differentiation by targeting mitotic clonal expansion and arresting the cell cycle in the G1 phase of the cell cycle. It improved the dyslipidemic condition in HFD-fed hamsters by decreasing the body weight, fat mass, eWAT weight and improving the serum lipid profile. Overall, PLH has been found as a potential drug candidate and a pharmacophore for combating metabolic disorders including obesity and dyslipidemia.

Pyrroloquinoline quinone (PQQ) improves pulmonary hypertension by regulating mitochondrial and metabolic functions.

Excessive proliferation of pulmonary artery smooth muscle cells (PASMCs) and endothelial cells (PAECs), inflammation, as well as mitochondrial and metabolic dysregulation, contributes to the development of pulmonary hypertension (PH). Pyrroloquinoline quinone (PQQ), a potent natural antioxidant with anti-diabetic, neuroprotective, and cardioprotective properties, is known to promote mitochondrial biogenesis. However, its effect on cellular proliferation, apoptosis resistance, mitochondrial and metabolic alterations associated with PH remains unexplored. The current study was designed to investigate the effect of PQQ in the treatment of PH. Human pulmonary artery smooth muscle cells (HPASMCs), endothelial cells (PAECs), and primary cultured cardiomyocytes were subjected to hypoxia to induce PH-like phenotype. Furthermore, Sprague Dawley (SD) rats injected with monocrotaline (MCT) (60 mg/kg, SC, once) progressively developed pulmonary hypertension. PQQ treatment (2 mg/kg, PO, for 35 days) attenuated cellular proliferation and promoted apoptosis via a mitochondrial-dependent pathway. Furthermore, PQQ treatment in HPASMCs prevented mitochondrial and metabolic dysfunctions, improved mitochondrial bioenergetics while preserving respiratory complexes, and reduced insulin resistance. In addition, PQQ treatment (preventive and curative) significantly attenuated the increase in right ventricle pressure and hypertrophy as well as reduced endothelial dysfunction and pulmonary artery remodeling in MCT-treated rats. PQQ also prevented cardiac fibrosis and improved cardiac functions as well as reduced inflammation in MCT-treated rats. Altogether, the above findings demonstrate that PQQ can attenuate mitochondrial as well as metabolic abnormalities in PASMCs and also prevent the development of PH in MCT treated rats; hence PQQ may act as a potential therapeutic agent for the treatment of PH.

Galectin-3 S-glutathionylation regulates its effect on adipocyte insulin signaling.

Protein-S-glutathionylation promotes redox signaling in physiological and oxidative distress conditions. Galectin-3 (Gal-3) promotes insulin resistance by down-regulating adipocyte insulin signaling, however, its S-glutathionylation and significance is not known. In this context, we report reversible S-glutathionylation of Gal-3. Site-directed mutagenesis established Gal-3 Cys187 as the putative S-glutathionylation site. Glutathionylated Gal-3 prevents Gal-3(WT)-Insulin Receptor interaction and facilitates insulin-induced murine adipocyte p-IRS1(tyr895) and p-AKT(ser473) signaling and glucose uptake in a Gal-3 Cys187 glutathionylation dependent manner in murine adipocytes, as assessed by Western blotting and 2-NBDG uptake assay respectively. Pre-glutathionylated Gal-3 at Cys187 resisted irreversible oxidation by H2O2. M2 macrophages showed enhanced Gal-3 S-glutathionylation when compared to M1 phenotype. Serum and stromal vascular fraction (SVF) isolated from control mice showed increased Gal-3 S-glutathionylation as compared to db/db mice. A significant increase in Gal-3 S-glutathionylation was observed in metformin-treated db/db mice when compared to db/db mice alone. Similar to murine, enhanced Gal-3 S-glutathionylation is observed in primary human monocyte derived M2 macrophages when compared to the M1 macrophage phenotype and Gal-3 regulates primary human adipocyte insulin signaling in a glutathionylation dependent manner. Collectively, we identified Gal-3 S-glutathionylation as a protective phenomenon, which relieves its inhibitory effect on adipocyte insulin signaling.

An Imaging and Computational Algorithm for Efficient Identification and Quantification of Neutrophil Extracellular Traps.

Neutrophil extracellular traps (NETs) are associated with multiple disease pathologies including sepsis, asthma, rheumatoid arthritis, cancer, systemic lupus erythematosus, acute respiratory distress syndrome, and COVID-19. NETs, being a disintegrated death form, suffered inconsistency in their identification, nomenclature, and quantifications that hindered therapeutic approaches using NETs as a target. Multiple strategies including microscopy, ELISA, immunoblotting, flow cytometry, and image-stream-based methods have exhibited drawbacks such as being subjective, non-specific, error-prone, and not being high throughput, and thus demand the development of innovative and efficient approaches for their analyses. Here, we established an imaging and computational algorithm using high content screening (HCS)-cellomics platform that aid in easy, rapid, and specific detection as well as analyses of NETs. This method employed membrane-permeable and impermeable DNA dyes in situ to identify NET-forming cells. Automated algorithm-driven single-cell analysis of change in nuclear morphology, increase in nuclear area, and change in intensities provided precise detection of NET-forming cells and eliminated user bias with other cell death modalities. Further combination with Annexin V staining in situ detected specific death pathway, e.g., apoptosis, and thus, discriminated between NETs, apoptosis, and necrosis. Our approach does not utilize fixation and permeabilization steps that disturb NETs, and thus, allows the time-dependent monitoring of NETs. Together, this specific imaging-based high throughput method for NETs analyses may provide a good platform for the discovery of potential inhibitors of NET formation and/or agents to modulate neutrophil death, e.g., NETosis-apoptosis switch, as an alternative strategy to enhance the resolution of inflammation.

Coelogin ameliorates metabolic dyshomeostasis by regulating adipogenesis and enhancing energy expenditure in adipose tissue.

Obesity and associated metabolic disorders are heading up with an alarming rate in developing nations. One of highly sought solution for metabolic disorders is to identify natural molecule with an ability to reduce obesity and increase insulin sensitivity. Coelogin (CLN) is a phenanthrene derivative isolated from the ethanolic extract of Coelogyne cristata. In our constant efforts to identify novel anti-dyslipidemic and anti-adipogenic compounds using CFPMA (common feature pharmacophore model using known anti-adipogenic compounds) model, predicted possible anti-adipogenic activity of CLN. In vitro results showed significant inhibition of adipogenesis in 3T3-L1 and C3H10T1/2 cell by CLN. It arrests the cell cycle in G1 phase of interphase and inhibits mitotic clonal expansion to regulate adipogenesis. CLN elicits insulin sensitizing effect in mature adipocytes. During extracellular flux assessment studies, it increases oxidative respiration and energy expenditure in adipocytes. In vivo, CLN reversed HFD-induced dyslipidemia as well as insulin resistance in C57BL/6 mice. It promoted the expression of genes involved in improved mitochondrial function and fatty acid oxidation in eWAT. CLN restored energy expenditure and increased the capacity of energy utilization in HFD fed mice. Taken together, the study indicated beneficial effects of CLN in combating obesity-associated metabolic complications.

Role of brown adipose tissue in modulating adipose tissue inflammation and insulin resistance in high-fat diet fed mice.

Obesity results in the chronic activation of innate immune system and subsequently sets in diabetes. Aim of the study was to investigate the immunometabolic role of brown adipose tissue (BAT) in the obesity. We performed both BAT transplantation as well as extirpation experiments in the mouse model of high-fat diet (HFD)-induced obesity. We carried out immune cell profiling in the stromal vascular fraction (SVF) isolated from epididymal white adipose tissue (eWAT). BAT transplantation reversed HFD-induced increase in body weight gain and insulin resistance without altering diet intake. Importantly, BAT transplantation attenuated the obesity-associated adipose tissue inflammation in terms of decreased pro-inflammatory M1-macrophages, cytotoxic CD8a T-cells and restored anti-inflammatory regulatory T-cells (Tregs) in the eWAT. BAT transplantation also improved endogenous BAT activity by elevating protein expression of browning markers (UCP-1, PRDM16 and PGC1α) in it. In addition, BAT transplantation promoted the eWAT expression of various genes involved in fatty acid oxidation (such as Elvol3 and Tfam,). In contrast, extirpation of the interscapular BAT exacerbated HFD-induced obesity, insulin resistance and adipose tissue inflammation (by increasing M1 macrophages, CD8a T-cell and decreasing Tregs in eWAT). Taken together, our results suggested an important role of BAT in combating obesity-associated metabolic complications. These results open a novel therapeutic option to target obesity and related metabolic disorders like type 2 diabetes.

Chronic hyperinsulinemia induced miR-27b is linked to adipocyte insulin resistance by targeting insulin receptor.

Defect in insulin signaling leads to the development of insulin resistance followed by type 2 diabetes. Exploiting our previously developed physiological chronic hyperinsulinemia (CI)-mediated insulin resistance (IR) model, we wanted to understand how miRNAs contribute to the development of IR. Amongst the identified and validate miRNAs, the expression of miR-27b was found to be highly upregulated during CI-induced IR in 3T3-L1 adipocytes. We also validated the expression of miR-27b in CI-induced IR in human mesenchymal stem cell (hMSC)-derived adipocytes and in vivo high fat diet (HFD)-induced IR mice model. Bioinformatics target prediction softwares and luciferase reporter assay identified insulin receptor (INSR) as one of a prime target of miR-27b. Lentiviral mediated overexpression of miR-27b impairs insulin signaling by modulating INSR expression that in turn led to decreased glucose uptake in both 3T3-L1 and hMSC-derived adipocytes. Conversely, inhibition of miR-27b reversed CI-mediated suppression of target protein INSR and improved phosphorylation of Akt, a nodal protein of insulin signaling that is impaired by CI treatment. Lentiviral mediated overexpression of miR-27b in in vivo C57BL/6 mice impaired whole body glucose tolerance and adipose tissue insulin sensitivity. Furthermore, inhibition of miR-27b in HFD-induced insulin resistance mice model improved glucose tolerance and adipose tissue insulin sensitivity by increasing the expression of its target gene INSR in eWAT. Thus, our results indicate that miR-27b functions as a prime modulator of CI-induced IR via regulating the expression of INSR. KEY MESSAGES: miR-27b is upregulated in different in vitro and in vivo models of insulin resistance. miR-27b directly suppresses the expression of INSR by targeting 3'UTR of INSR. Modulation of miR-27b expression regulates insulin sensitivity by targeting INSR.

Aegeline inspired synthesis of novel amino alcohol and thiazolidinedione hybrids with antiadipogenic activity in 3T3-L1 cells.

Excess adiposity is a hallmark of obesity, which is caused due to an imbalance between energy intake and energy consumed. Obesity is often associated with several metabolic disorders like dyslipidemia, cardiovascular diseases and type 2 diabetes. Earlier, our group had reported natural product Aegeline (amino-alcohol) isolated from the plant Aegle marmelos as an anti-diabetic and anti-dyslipidemic compound. With this background, we synthesized a series of novel amino alcohol and thiazolidinedione hybrid molecules and studied their antiadipogenic activity. As a result, we have identified a potent hybrid compound 12c as an inhibitor of adipocyte differentiation. The compound 12c inhibits lipid accumulation and adipogenesis in 3T3-L1 preadipocyte cell line. Exposure of compound 12c blocks mitotic clonal expansion and arrests cells in S-phase of cell cycle. Detailed analysis showed that compound 12c decreases expression of two major transcription factors that are involved in adipocyte differentiation, PPARγ, C/EBPα, and other adipogenesis associated genes like aP2 and FAS. Thus, we concluded that compound 12c shows potential ability to inhibit adipocyte differentiation which can be used therapeutically for the treatment of obesity and its associated metabolic disorders.

Ecliptal, a promising natural lead isolated from Eclipta alba modulates adipocyte function and ameliorates metabolic syndrome.

A swift increase has been observed in the number of individuals with metabolic syndrome worldwide. A number of natural compounds have been identified towards combating metabolic syndrome. Adding to this premise, here we report the pleiotropic activities of Ecliptal (EC); a natural compound isolated from the herb Eclipta alba. Administration of EC was shown to have prominent anti-adipogenic effects in 3T3-L1 and hMSC derived adipocytes. It was shown to activate Wnt-pathway and alter AKT signaling. Additionally, it caused cell cycle arrest and inhibited mitotic clonal expansion. EC treatment augmented mitochondrial biogenesis as well as function as estimated by expression of PGC1α, UCP-1, mitochondrial complexes and estimation of oxygen consumption rate. EC also reduced LPS-induced inflammation and tunicamycin induced ER stress. Further, EC enhanced insulin sensitivity by increasing AKT phosphorylation, inhibiting PKCα/ßII phosphorylation and reducing leptin/adiponectin ratio. Finally, EC administration in Syrian golden hamsters was shown to have potent anti-dyslipidemic effects. Cumulatively, encompassing pleiotropic activities of EC, it could prove to be a potential drug candidate against obesity, insulin resistance and related metabolic syndrome.

Ethyl acetate fraction of Eclipta alba: a potential phytopharmaceutical targeting adipocyte differentiation.

Natural products have always fascinated mankind for their miraculous properties. Eclipta alba (E. alba), a medicinal herb has long been used in traditional medicine for curing several pathologies. It has been shown to have anti-diabetic effect as well as hepato-protective activity. Here, in order to address metabolic derangements, the study was designed to evaluate the efficacy of E. alba and its fractions in adipogenesis inhibition and dyslipidemia. Of the crude extract and fractions screened, ethyl acetate fraction of E. alba inhibited adipocyte differentiation in 3T3-L1 pre-adipocytes and hMSC derived adipocytes. It inhibited mitotic clonal expansion and caused cell cycle arrest in G1 and S phase as suggested by western blot analysis and flow cytometry. It was also shown to have lipolytic effects. Oral administration of ethyl acetate fraction of E. alba to hamsters unveiled its anti-adipogenic as well as anti-dyslipidemic activity in-vivo. Mass spectrometry analysis of ethyl acetate fraction confirmed the presence of several bioactive components, projecting it as an effective phytopharmaceutical agent. In conclusion, ethyl acetate fraction of E. alba possesses potent anti-adipogenic as well as anti-dyslipidemic activity and could be projected as an herbal formulation towards obesity.

Curcumin-3,4-Dichloro Phenyl Pyrazole (CDPP) overcomes curcumin's low bioavailability, inhibits adipogenesis and ameliorates dyslipidemia by activating reverse cholesterol transport.

BACKGROUND: Adipocyte dysfunction, obesity and associated metabolic disorders are of prime healthcare concern worldwide. Among available medications, natural products and inspired molecules hold 40% space in clinically prescribed medicines. In queue, this study overcomes the drawback of curcumin's low bioavailability with potent anti-adipogenic and anti-dyslipidemic activity. METHODS: To evaluate the role of CDPP on adipocyte differentiation, 3T3-L1 adipocytes were used as an in-vitro model. Flow cytometry was performed for cell cycle analysis. Syrian golden hamsters were used to study pharmacokinetic profile and dyslipidemic activity exhibited by CDPP. RESULT: CDPP was found to be a potent inhibitor of adipogenesis in-vitro. It blocked mitotic clonal expansion by causing cell cycle arrest. CDPP showed marked improvement in gastrointestinal stability and bioavailability in-vivo as compared to curcumin. Administration of CDPP (100mg/kg) significantly improved HFD induced dyslipidemic profile in hamsters and activated reverse cholesterol transport machinery. CONCLUSION: CDPP could be used as a potential drug candidate against adipogenesis and dyslipidemia with enhanced gastrointestinal stability and bioavailability.

Chronic hyper-leptinemia induces insulin signaling disruption in adipocytes: Implications of NOS2.

Leptin, following its discovery, has developed a formidable interest in the scientific community to delineate its contribution towards overall metabolic homeostasis. Contradictory reports have been published on leptin administration effects on whole body insulin sensitivity. Following late reports, we surveyed human serum leptin levels along with other metabolic parameters including BMI and HOMA-IR. We found a positive correlation between leptin levels and insulin resistance parameters. Considering the presence of the long form of leptin receptor on adipocytes, we explored the effects of chronic physiological hyper-leptinemic exposure on adipocyte insulin sensitivity. Chronic leptin (50ng/ml) treatment in 3T3-L1 adipocytes decreased insulin-induced phosphorylation of nodal insulin signaling proteins along with reduced glucose uptake. Metabolic flux studies indicated mitochondrial dysfunction and reduced oxygen consumption rate. Leptin treatment also increased both cellular and mitochondrial superoxide levels concomitant to increased expression of nitric oxide synthase-2 (NOS2). Further, pharmacological depletion of NOS2 reversed leptin mediated effects on insulin signaling. In-vivo implantation of leptin osmotic pumps in C57BL/6 mice also decreased insulin responsiveness. Interestingly, these effects were lacking in NOS2 knockout strain. In conclusion, our studies put forward a potential link between leptin and adipocyte insulin responsiveness in an NOS2 dependent manner.

16-Dehydropregnenolone lowers serum cholesterol by up-regulation of CYP7A1 in hyperlipidemic male hamsters.

16-Dehydropregnenolone (DHP) has been developed and patented as a promising antihyperlipidemic agent by CSIR-Central Drug Research Institute (CSIR-CDRI), India. Although DHP is implicated in controlling cholesterol homeostasis, the mechanism underlying its pharmacological effect in hyperlipidemic disease models is poorly understood. In the present study, we postulated that DHP lowers serum lipids through regulating the key hepatic genes accountable for cholesterol metabolism. The hypothesis was tested on golden Syrian hamsters fed with high-fat diet (HFD) following oral administration of DHP at a dose of 72mg/kg body weight for a period of one week. The serum total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and total bile acids (TBA) in feces were measured. Real time comparative gene expression studies were performed for CYP7A1, LXRα and PPARα level in liver tissue of hamsters. The results revealed that the DHP profoundly decreased the levels of serum TC, TG, LDL-C and atherogenic index (AI), whilst elevated the HDL-C/TC ratio. Besides, DHP exhibited an anti-hyperlipidemic effect in the HFD induced hyperlipidemic hamsters by means of: (1) up-regulating the gene expression of CYP7A1 encoded cholesterol 7α-hydroxylase, that promotes the catabolism of cholesterol to bile acid; (2) inducing the gene expression of transcription factors LXRα and PPARα; (3) increasing the TBA excretion through feces. Collectively, the findings presented confer the hypolipidemic activity of DHP via up-regulation of hepatic CYP7A1 pathway that promotes cholesterol-to-bile acid conversion and bile acid excretion.

A clerodane diterpene inhibit adipogenesis by cell cycle arrest and ameliorate obesity in C57BL/6 mice.

A clerodane diterpene, 16α-Hydroxycleroda-3, 13 (14) Z-dien-15, 16-olide (compound 1) isolated from Polyalthia longifolia had previously been reported as a new structural class of HMG-CoA reductase inhibitor apart from statins. Statins are known to be anti-adipogenic in nature. The distant structural similarity between compound 1 and lovastatin (polyketide class of compound) prompted us to investigate effects of diterpene compound 1 on adipogenesis and thereby obesity. High content microscopy proved diterpene compound 1 exhibits better anti-adipogenic activity and less toxicity in differentiating adipocytes. Moreover, it reduced expression levels of PPARγ, C/EBPα and GLUT4 during differentiation in a time and concentration dependent manner. Diterpene compound 1 during early differentiation reduced MDI induced-Akt/mTOR phosphorylation and expression of cell cycle proteins, and thereby halted mitotic clonal expansion, the decisive factor in early adipogenesis. Further, its anti-adipogenic activity was validated in murine mesenchymal cell-line C3H10T1/2 and human mesenchymal stem cell models of adipogenic differentiation. When compound 1 was administered along with HFD, for another 8 weeks in 2 month HFD fed overweight mice (with BMI > 30 and impaired glucose tolerance), it attenuated weight gain and epididymal fat accumulation. It improved body glucose tolerance, reduced HFD induced increase in total cholesterol and leptin/adiponectin ratio. All these effects were comparable with standard anti-obesity drug Orlistat with added edge of potently decreasing circulating triglyceride levels comparable with normal chow fed group. Histological analysis shows that compound 1 inhibit adipocyte hypertrophy and decreased steatosis in hepatocytes. Both in vivo and in vitro results demonstrate a potential value of compound 1 as a novel anti-adipogenic and anti-obesity agent.

Identification of novel PTP1B inhibitors by pharmacophore based virtual screening, scaffold hopping and docking.

Design and synthesis of protein tyrosine phosphatases-1B (PTP1B) inhibitors are important for the drugs targeted to treat diabetes and obesity. The pharmacophore modeling, docking and scaffold hopping techniques have been applied to discover the novel PTP1B inhibitors. The ten prioritized compounds (115-119, 120-121, 127, 130-131) from the library of 86 compounds were synthesized and found positive in the micro molar range for PTP1B in-vitro inhibitory assays as compared to Suramin (IC50 9.5 µM). Among these five active compounds (115-119) were tested in STZ-s induced diabetic rat model and the most active compound 115 in this test, was further tested in C57BL/KsJ-db/db mice where it significantly improved OGTT along with the fasting and random blood glucose level. The treatment by the compound 115 significantly improved the insulin resistance and insulin signaling by restoring the insulin level and normalizing the serum lipid profile. Compound 115 also augmented the insulin action by modulating the expression of genes involved in insulin signaling like IRS 1-2, PI3K, PTPN1, Akt2, AMPK and PPAR-α. Western blot analysis of both skeletal muscle and liver demonstrated that proteins and intermediate enzymes of insulin signaling were also increased as compared to control group. The compound 115 was also investigated for anti-adipogenic effect on 3T3L-1 cells. The compound 115 inhibited MDI induced lipid accumulation in a dose-dependent manner. The oral bioavailability of compound 115 was ∼10.29% after 30 mg/kg oral dosing assessed in rat.

Adipocyte transdifferentiation and its molecular targets.

According to the World Health Organization obesity is defined as the excessive accumulation of fat, which increases risk of other metabolic disorders such as insulin resistance, dyslipidemia, hypertension, cardiovascular diseases, etc. There are two types of adipose tissue, white and brown adipose tissue (BAT) and the latter has recently gathered interest of the scientific community. Discovery of BAT has opened avenues for a new therapeutic strategy for the treatment of obesity and related metabolic syndrome. BAT utilizes accumulated fatty acids for energy expenditure; hence it is seen as one of the possible alternates to the current treatment. Moreover, browning of white adipocyte on exposure to cold, as well as with some of the pharmacological agents presents exciting outcomes and indicates the feasibility of transdifferentiation. A better understanding of molecular pathways and differentiation factors, those that play a key role in transdifferentiation are of extreme importance in designing novel strategies for the treatment of obesity and associated metabolic disorders.

Pharmacokinetic and metabolism studies of rohitukine in rats by high performance liquid-chromatography with tandem mass spectrometry.

A sensitive, selective, and rapid high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed for the quantification of rohitukine in rat plasma. HPLC was performed using a Symmetry-Shield C18 (5 µ, 4.6 × 150 mm) column, and isocratic elution with ammonium acetate buffer (pH4; 10 mM):methanol (08:92, v/v) at a flow rate of 0.6 mL/min. Sample clean-up involved solid phase extraction (SPE) of analyte and internal standard (phenacetin) from 100 µL plasma. The parent→product ion transitions (MRM) for analyte and IS were 306.1→245.1 m/z and 180.1→138.1 m/z respectively, and were monitored on a triple quadrupole mass spectrometer, operating in positive ion mode. The method was validated across the dynamic concentration range of 5-500 ng/mL for rohitukine, with a fast run time of 4.5 min. The analytical method measured concentrations of rohitukine with accuracy (% bias) of <±10% and precision (% RSD) of <±12%. Rohitukine was stable during the battery of stability studies viz., bench-top, auto-sampler, freeze/thaw cycles and 30 days of storage in a freezer at -70±10°C. Finally, the applicability of this assay has been successfully demonstrated in vivo pharmacokinetic and in vitro metabolism studies in Sprague-Dawley rat. This method will therefore be highly useful for future preclinical and clinical pharmacokinetic studies of rohitukine.