Natural Compound 3β,7β,25-trihydroxycucurbita-5,23(E)-dien-19-al from Momordica charantia Acts as PPARγ Ligand

Momordica charantia is a popular vegetable associated with effective complementary and alternative diabetes management in some parts of the world. However, the molecular mechanism is less commonly investigated. In this study, we investigated the association between a major cucurbitane triterpenoid isolated from M. charantia, 3β,7β,25-trihydroxycucurbita-5,23(E)-dien-19-al (THCB) and peroxisome proliferator activated receptor gamma (PPARγ) activation and its related activities using cell culture and molecular biology techniques. In this study, we report on both M. charantia fruit crude extract and THCB in driving the luciferase activity of Peroxisome Proliferator Response Element, associated with PPARγ activation. Other than that, THCB also induced adipocyte differentiation at far less intensity as compared to the full agonist rosiglitazone. In conjunction, THCB treatment on adipocytes also resulted in upregulation of PPAR gamma target genes expression; AP2, adiponectin, LPL and CD34 at a lower magnitude compared to rosiglitazone’s induction. THCB also induced glucose uptake into muscle cells and the mechanism is via Glut4 translocation to the cell membrane. In conclusion, THCB acts as one of the many components in M. charantia to induce hypoglycaemic effect by acting as PPARγ ligand and inducing glucose uptake activity in the muscles by means of Glut4 translocation.     

F-box only protein 9 is an E3 ubiquitin ligase of PPARγ

Peroxisome proliferator-activated receptor gamma (PPARγ) is a critical regulator of carbohydrate and lipid metabolism, adipocyte differentiation and inflammatory response. Post-translational modification of PPARγ and its degradation involve several pathways, including the ubiquitin–proteasome system. Here, we identified F-box only protein 9 (FBXO9) as an E3 ubiquitin ligase of PPARγ. We screened interacting partners of PPARγ using immunoprecipitation and mass spectrometric analysis and identified FBXO9 as an E3 ubiquitin ligase of PPARγ. FBXO9 directly interacted with PPARγ through the activation function-1 domain and ligand-binding domain. FBXO9 decreased the protein stability of PPARγ through induction of ubiquitination. We found that the F-box motif of FBXO9 was required for its ubiquitination function. The activity of PPARγ was significantly decreased by FBXO9 overexpression. Furthermore, FBXO9 overexpression in 3T3-L1 adipocytes resulted in decreased levels of endogenous PPARγ and suppression of adipogenesis. These results suggest that FBXO9 is an important enzyme that regulates the stability and activity of PPARγ through ubiquitination.     

Pyruvate dehydrogenase kinase 1 and 2 deficiency reduces high-fat diet-induced hypertrophic obesity and inhibits the differentiation of preadipocytes into mature adipocytes

Obesity is now recognized as a disease. This study revealed a novel role for pyruvate dehydrogenase kinase (PDK) in diet-induced hypertrophic obesity. Mice with global or adipose tissue-specific PDK2 deficiency were protected against diet-induced obesity. The weight of adipose tissues and the size of adipocytes were reduced. Adipocyte-specific PDK2 deficiency slightly increased insulin sensitivity in HFD-fed mice. In studies with 3T3-L1 preadipocytes, PDK2 and PDK1 expression was strongly increased during adipogenesis. Evidence was found for epigenetic induction of both PDK1 and PDK2. Gain- and loss-of-function studies with 3T3-L1 cells revealed a critical role for PDK1/2 in adipocyte differentiation and lipid accumulation. PDK1/2 induction during differentiation was also accompanied by increased expression of hypoxia-inducible factor-1α (HIF1α) and enhanced lactate production, both of which were absent in the context of PDK1/2 deficiency. Exogenous lactate supplementation increased the stability of HIF1α and promoted adipogenesis. PDK1/2 overexpression-mediated adipogenesis was abolished by HIF1α inhibition, suggesting a role for the PDK-lactate-HIF1α axis during adipogenesis. In human adipose tissue, the expression of PDK1/2 was positively correlated with that of the adipogenic marker PPARγ and inversely correlated with obesity. Similarly, PDK1/2 expression in mouse adipose tissue was decreased by chronic high-fat diet feeding. We conclude that PDK1 and 2 are novel regulators of adipogenesis that play critical roles in obesity.Subject terms: Mechanisms of disease, Obesity     

Anti-Inflammatory and Anti-Diabetic Effect of Black Soybean Anthocyanins: Data from a Dual Cooperative Cellular System

Obesity is characterized by elevated infiltration of macrophages into adipose tissue, leading to the development of insulin resistance. The black soybean seed coat is a rich source of anthocyanins with antioxidative and anti-inflammatory activities. This study investigated the effects of black soybean anthocyanin extract (BSAn) on obesity-induced oxidative stress, the inflammatory response, and insulin resistance in a coculture system of hypertrophied 3T3-L1 adipocytes and RAW264 macrophages. Coculture of adipocytes with macrophages increased the production of reactive oxygen species and inflammatory mediators and cytokines (NO, MCP-1, PGE₂, TNFα, and IL-6) and the release of free fatty acids but reduced anti-inflammatory adiponectin secretion. BSAn treatment (12.5, 25, 50, and 100 μg/mL) alleviated the coculture-induced changes (p < 0.001) and inhibited coculture-induced activation of JNK and ERK signaling (p < 0.01). BSAn also blocked the migration of RAW264.7 macrophages toward 3T3-L1 adipocytes. In addition, treatment with BSAn increased PPARγ expression and glucose uptake in response to insulin in hypertrophied 3T3-L1 adipocyte and RAW264.7 macrophage coculture (p < 0.01). These results demonstrate that BSAn attenuates inflammatory responses and improves adipocyte metabolic function in the coculture of hypertrophied 3T3-L1 adipocytes and RAW264.7 macrophages, suggesting the effectiveness of BSAn for obesity-induced insulin resistance.     

Opuntia dillenii Haw. Polysaccharide Promotes Cholesterol Efflux in THP-1-Derived Foam Cells via the PPARγ-LXRα Signaling Pathway

There is increasing evidence supporting a role for enhanced macrophage cholesterol efflux in ameliorating atherosclerosis. Opuntia dillenii Haw. polysaccharide (ODP-Ia), the most important functional component obtained from Opuntia dillenii Haw. stem, has anti-atherosclerosis effects. Therefore, we propose that ODP-Ia could promote cholesterol efflux via the PPARγ-LXRα signaling pathway. In this study, THP-1 foam cells derived from macrophages were treated with different concentrations of ODP-Ia, GGPP (antagonist of LXRα) and GW9662 (antagonist of PPARγ), with or without 15 nmol ODP-Ia. The total cholesterol content in the cells was measured. The mRNA of ABCA1, ABCG1, PPARγ, LXRα and their protein levels in the foam cells were detected by RT–PCR and Western blot, respectively. The results showed that ODP-Ia plays a role in significantly promoting cholesterol efflux (p < 0.05) by upregulating the expression of ABCA1, ABCG1, SR-BI, PPARγ, PPARα and LXRα. Meanwhile, PPARγ and LXRα antagonists dramatically interfered the cholesterol efflux mediated by ODP-Ia (p < 0.05) and dramatically inhibited the upregulating effect of ODP-Ia on the expression of PPARγ, LXRα, ABCA1 and ABCG1 at both protein and mRNA levels (p < 0.05). In conclusion, ODP-Ia promotes cholesterol efflux in the foam cells through activating the PPARγ-LXRα signaling pathway. This bioactivity suggested that ODP-Ia may be of benefit in treating atherosclerosis.     

Ameliorative Effect of Ocimum forskolei Benth on Diabetic,Apoptotic, and Adipogenic Biomarkers of Diabetic Rats and 3T3-L1 Fibroblasts Assisted by In Silico Approach

Diabetes mellitus (DM) is a complicated condition that is accompanied by a plethora of metabolic symptoms, including disturbed serum glucose and lipid profiles. Several herbs are reputed as traditional medicine to improve DM. The current study was designed to explore the chemical composition and possible ameliorative effects of Ocimum forskolei on blood glucose and lipid profile in high-fat diet/streptozotocin-induced diabetic rats and in 3T3-L1 cell lines as a first report of its bioactivity. Histopathological study of pancreatic and adipose tissues was performed in control and treatment groups, along with quantification of glucose and lipid profiles and the assessment of NF-κB, cleaved caspase-3, BAX, and BCL2 markers in rat pancreatic tissue. Glucose uptake, adipogenic markers, DGAT1, CEBP/α, and PPARγ levels were evaluated in the 3T3-L1 cell line. Hesperidin was isolated from total methanol extract (TME). TME and hesperidin significantly controlled the glucose and lipid profile in DM rats. Glibenclamide was used as a positive control. Histopathological assessment showed that TME and hesperidin averted necrosis and infiltration in pancreatic tissues, and led to a substantial improvement in the cellular structure of adipose tissue. TME and hesperidin distinctly diminished the mRNA and protein expression of NF-κB, cleaved caspase-3, and BAX, and increased BCL2 expression (reflecting its protective and antiapoptotic actions). Interestingly, TME and hesperidin reduced glucose uptake and oxidative lipid accumulation in the 3T3-L1 cell line. TME and hesperidin reduced DGAT1, CEBP/α, and PPARγ mRNA and protein expression in 3T3-L1 cells. Moreover, docking studies supported the results via deep interaction of hesperidin with the tested biomarkers. Taken together, the current study demonstrates Ocimum forskolei and hesperidin as possible candidates for treating diabetes mellitus.     

Inhibition of Lipid Accumulation and Adipokine Levels in Maturing Adipocytes by Bauhinia rufescens (Lam.) Stem Bark Extract Loaded Titanium Oxide Nanoparticles

The present study reports a cost-effective, environmentally friendly method to increase the bioavailability and bio-efficacy of B. rufescens stem bark extract in the biological system via functional modification as B. rufescens stem bark nanoparticles (BR-TO₂-NPs). The biosynthesis of BR- -NPs was confirmed by UV-visible (UV-vis) and Fourier-transform infrared (FT-IR) spectroscopy, transmission electron microscopy (TEM), and X-ray diffraction analyses. The shifts in FT-IR stretching vibrations of carboxylic and nitro groups (1615 cm⁻¹), the O–H of phenolics or carboxylic acids (3405 cm⁻¹), alkanes, and alkyne groups (2925 and 2224 cm⁻¹) of the plant extract and lattice (455) indicated successful biosynthesis of BR- -NPs. Compared with the stem bark extract, 40 ng/dL dose of BR- -NPs led to a reduction in adipogenesis and an increase in mitochondrial biogenesis-related gene expressions, adiponectin-R1, PPARγC1α, UCP-1, and PRDM16, in maturing-adipocytes. This confirmed the intracellular uptake, bioavailability, and bio-efficiency of BR-TiO₂-NPs. The lipid-lowering capacity of BR-TiO₂-NPs effectively inhibited the metabolic inflammation-related gene markers, IL-6, TNF-α, LTB4-R, and Nf-κb. Further, BR-TiO₂-NPs stimulating mitochondrial thermogenesis capacity was proven by the significantly enhanced CREB-1 and AMPK protein levels in adipocytes. In conclusion, BR-TiO₂-NPs effectively inhibited lipid accumulation and proinflammatory adipokine levels in maturing adipocytes; it may help to overcome obesity-associated comorbidities.     

Hypoglycemic and Antioxidant Properties of Extracts and Fractions from Polygoni Avicularis Herba

Our research focused on the hypoglycemic capability and the possible mechanisms of extract and fractions from Polygoni Avicularis Herba (PAH) based on α-glucosidase, α-amylase inhibition assays, glucose uptake experiment, HPLC-MS analysis, and molecular docking experiment. In addition, DPPH, ABTS, and FRAP assays were used for determining the antioxidant capability. The results of total flavonoids and phenolics contents showed that ethyl acetate fraction (EAF) possessed the highest flavonoids and phenolics with values of 159.7 ± 2.5 mg rutin equivalents/g and 107.6 ± 2.0 mg galic acid equivalents/g, respectively. The results of in vitro hypoglycemic activity showed that all samples had effective α-glucosidase inhibition capacities, and EAF possessed the best inhibitory effect with IC₅₀ value of 1.58 ± 0.24 μg/mL. In addition, n-butanol fraction (NBF) significantly promoted the glucose uptake rate of 3T3-L1 adipocytes. HPLC-MS analysis and molecular docking results proved the interactions between candidates and α-glucosidase. The results of antioxidation capacities showed that EAF possessed the best antioxidation abilities with DPPH, ABTS, and FRAP. In summary, the hypoglycemic activity of PAH might be related to the inhibition of α-glucosidase (EAF > PEF > NBF) and the promotion of glucose uptake in 3T3-L1 adipocytes (NBF). Simultaneously, the antioxidation capacity of PAH might be related to the abundant contents of flavonoids and other phenolics (EAF > PEF > NBF).     

Ocimum basilicum L. Methanol Extract Enhances Mitochondrial Efficiency and Decreases Adipokine Levels in Maturing Adipocytes Which Regulate Macrophage Systemic Inflammation

Excessive storage of lipids in visceral or ectopic sites stimulates adipokine production, which attracts macrophages. This process determines the pro- and anti-inflammatory response regulation in adipose tissue during obesity-associated systemic inflammation. The present study aimed to identify the composition of Ocimum basilicum L. (basil) seed extract and to determine its bio-efficacy on adipocyte thermogenesis or fatty acid oxidation and inhibition of lipid accumulation and adipokine secretion. Ocimum basilicum L. seed methanol extract (BSME) was utilized to analyze the cytotoxicity vs. control; lipid accumulation assay (oil red O and Nile red staining), adipogenesis and mitochondrial-thermogenesis-related gene expression vs. vehicle control were analyzed by PCR assay. In addition, vehicle control and BSME-treated adipocytes condition media were collected and treated with lipopolysaccharide (LPS)-induced macrophage to identify the macrophage polarization. The results shown that the active components present in BSME did not produce significant cytotoxicity in preadipocytes or macrophages in the MTT assay. Furthermore, oil red O and Nile red staining assay confirmed that 80 and 160 μg/dL concentrations of BSME effectively arrested lipid accumulation and inhibited adipocyte maturation, when compared with tea polyphenols. Gene expression level of adipocyte hyperplasia (CEBPα, PPARγ) and lipogenesis (LPL)-related genes have been significantly (p ≤ 0.05) downregulated, and mitochondrial-thermogenesis-associated genes (PPARγc1α, UCP-1, prdm16) have been significantly (p ≤ 0.001) upregulated. The BSME-treated, maturing, adipocyte-secreted proteins were detected with a decreased protein level of leptin, TNF-α, IL-6 and STAT-6, which are associated with insulin resistance and macrophage recruitment. The “LPS-stimulated macrophage” treated with “BSME-treated adipocytes condition media”, shown with significant (p ≤ 0.001) decrease in metabolic-inflammation-related proteins—such as PGE-2, MCP-1, TNF-α and NF-κB—were majorly associated with the development of foam cell formation and progression of atherosclerotic lesion. The present findings concluded that the availability of active principles in basil seed effectively inhibit adipocyte hypertrophy, macrophage polarization, and the inflammation associated with insulin resistance and thrombosis development. Ocimum basilicum L. seed may be useful as a dietary supplement to enhance fatty acid oxidation, which aids in overcoming metabolic complications.     

Regulation of anti-inflammatory cytokines IL-10 and TGF-β in mouse dendritic cells through treatment with Clonorchis sinensis crude antigen

Dendritic cells (DCs), which are regarded as the most potent antigen-presenting cells, are involved in innate and adaptive immunity. Upon uptake of pathogens, DCs express cell surface markers and secrete cytokines. In this study, we analyzed production of cytokines and found that interleukin (IL)-10 and transforming growth factor (TGF)-β production significantly increased in bone marrow-derived DCs and a mouse DC line, DC2.4, after treatment with crude antigen (CA) from liver fluke, Clonorchis sinensis. However, expression patterns of several activation molecules did not change. In addition, following treatment of DC2.4 cells with antigen from the lung fluke, Paragonimus westermani, production of IL-10 and TGF-β significantly increased compared with groups treated with other parasite antigens, Spirometra erinacei plerocercoid CA and Echinococcus granulosus hydatid cystic fluid. We also found that treatment of DC2.4 cells with C. sinensis CA resulted in rapid and significant phosphorylation of extracellular signal-regulated kinase 1/2, a mitogen-activated protein kinase. Following treatment of DC2.4 cells with C. sinensis CA, treatment with an inhibitor specific to an extracellular signal-regulated kinase inhibited production of IL-10 and TGF-β. Our results suggest that CA from C. sinensis has a role in the anti-inflammatory function of DC cells by inducing IL-10 and TGF-β through activation of extracellular signal-regulated kinase 1/2.     

Flavonoids and Phenols,the Potential Anti-Diabetic Compounds from Bauhinia strychnifolia Craib. Stem.

Bioactive compounds from medicinal plants are good alternative treatments for T2DM. They are also sources of lead molecules that could lead to new drug discoveries. In this study, Bauhinia strychnifolia Craib. stem, a traditional Thai medicinal plant for detoxification, was extracted into five fractions, including crude extract, BsH, BsD, BsE, and BsW, by ethanolic maceration and sequential partition with hexane, dichloromethane, ethyl acetate, and water, respectively. Among these fractions, BsE contained the highest amounts of phenolics (620.67 mg GAE/g extract) and flavonoids (131.35 mg QE/g extract). BsE exhibited the maximum inhibitory activity against α-glucosidase (IC₅₀ 1.51 ± 0.01 µg/mL) and DPP-IV (IC₅₀ 2.62 ± 0.03 µg/mL), as well as dominantly promoting glucose uptake on 3T3-L1 adipocytes. Furthermore, the four compounds isolated from the BsE fraction, namely resveratrol, epicatechin, quercetin, and gallic acid, were identified. Quercetin demonstrated the highest inhibitory capacity against α-glucosidase (IC₅₀ 6.26 ± 0.36 µM) and DPP-IV (IC₅₀ 8.25 µM). In addition, quercetin prominently enhanced the glucose uptake stimulation effect on 3T3-L1 adipocytes. Altogether, we concluded that quercetin was probably the principal bioactive compound of the B. strychnifolia stem for anti-diabetic, and the flavonoid-rich fraction may be sufficiently potent to be an alternative treatment for blood sugar control.     

Activation of a Sweet Taste Receptor by Oleanane-Type Glycosides from Wisteria sinensis

The phytochemical study of Wisteria sinensis (Sims) DC. (Fabaceae), commonly known as the Chinese Wisteria, led to the isolation of seven oleanane-type glycosides from an aqueous-ethanolic extract of the roots. Among the seven isolated saponins, two have never been reported before: 3-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl-(1→2)-β-D-glucuronopyranosyl-22-O-acetylolean-12-ene-3β,16β,22β,30-tetrol, and 3-O-β-D-xylopyranosyl-(1→2)-β-D-glucuronopyranosylwistariasapogenol A. Based on the close structures between the saponins from W. sinensis, and the glycyrrhizin from licorice, the stimulation of the sweet taste receptor TAS1R2/TAS1R3 by these glycosides was evaluated.     

Bioactive Compounds from Lemon (Citrus limon) Extract Overcome TNF-α-Induced Insulin Resistance in Cultured Adipocytes

The consumption of plant-based food is important for health promotion, especially regarding the prevention and management of chronic diseases such as diabetes. We investigated the effects of a lemon extract (LE), containing ≥20.0% total flavanones and ≥1.0% total hydroxycinnamic acids, on insulin signaling in murine 3T3-L1 adipocytes treated with TNF-α, which was used to mimic in vitro the insulin resistance condition that characterizes diabetes mellitus. Our results showed LE increased PPARγ, GLUT4 and DGAT-1 levels, demonstrating the potential of this lemon extract in the management of insulin resistance conditions associated with TNF-α pathway activation. LE treatment further decreased the release of interleukin 6 (IL-6) and restored triglyceride synthesis, which is the main feature of a healthy adipocyte.     

Effects of Intra-BLA Administration of PPAR Antagonists on Formalin-Evoked Nociceptive Behaviour,Fear-Conditioned Analgesia,and Conditioned Fear in the Presence or Absence of Nociceptive Tone in Rats

There is evidence for the involvement of peroxisome proliferator-activated receptors (PPARs) in pain, cognition, and anxiety. However, their role in pain–fear interactions is unknown. The amygdala plays a key role in pain, conditioned fear, and fear-conditioned analgesia (FCA). We investigated the effects of intra-basolateral amygdala (BLA) administration of PPARα, PPARβ/δ, and PPARγ antagonists on nociceptive behaviour, FCA, and conditioned fear in the presence or absence of nociceptive tone. Male Sprague-Dawley (SD) rats received footshock (FC) or no footshock (NFC) in a conditioning arena. Twenty-three and a half hours later, rats received an intraplantar injection of formalin or saline and, 15 min later, intra-BLA microinjections of vehicle, PPARα (GW6471) PPARβ/δ (GSK0660), or PPARγ (GW9662) antagonists before arena re-exposure. Pain and fear-related behaviour were assessed, and neurotransmitters/endocannabinoids measured post-mortem. Intra-BLA administration of PPARα or PPARγ antagonists potentiated freezing in the presence of nociceptive tone. Blockade of all PPAR subtypes in the BLA increased freezing and BLA dopamine levels in NFC rats in the absence of nociceptive tone. Administration of intra-BLA PPARα and PPARγ antagonists increased levels of dopamine in the BLA compared with the vehicle-treated counterparts. In conclusion, PPARα and PPARγ in the BLA play a role in the expression or extinction of conditioned fear in the presence or absence of nociceptive tone.     

β-Sitosterol-D-Glucopyranoside Mimics Estrogenic Properties and Stimulates Glucose Utilization in Skeletal Muscle Cells

Estrogenic molecules have been reported to regulate glucose homeostasis and may be beneficial for diabetes management. Here, we investigated the estrogenic effect of β-sitosterol-3-O-D-glucopyranoside (BSD), isolated from the fruits of Cupressus sempervirens and monitored its ability to regulate glucose utilization in skeletal muscle cells. BSD stimulated ERE-mediated luciferase activity in both ERα and ERβ-ERE luc expression system with greater response through ERβ in HEK-293T cells, and induced the expression of estrogen-regulated genes in estrogen responsive MCF-7 cells. In silico docking and molecular interaction studies revealed the affinity and interaction of BSD with ERβ through hydrophobic interaction and hydrogen bond pairing. Furthermore, prolonged exposure of L6-GLUT4myc myotubes to BSD raised the glucose uptake under basal conditions without affecting the insulin-stimulated glucose uptake, the effect associated with enhanced translocation of GLUT4 to the cell periphery. The BSD-mediated biological response to increase GLUT4 translocation was obliterated by PI-3-K inhibitor wortmannin, and BSD significantly increased the phosphorylation of AKT (Ser-473). Moreover, BSD-induced GLUT4 translocation was prevented in the presence of fulvestrant. Our findings reveal the estrogenic activity of BSD to stimulate glucose utilization in skeletal muscle cells via PI-3K/AKT-dependent mechanism.     

Phloridzin Acts as an Inhibitor of Protein-Tyrosine Phosphatase MEG2 Relevant to Insulin Resistance

Inhibition of the megakaryocyte protein tyrosine phosphatase 2 (PTP-MEG2, also named PTPN9) activity has been shown to be a potential therapeutic strategy for the treatment of type 2 diabetes. Previously, we reported that PTP-MEG2 knockdown enhances adenosine monophosphate activated protein kinase (AMPK) phosphorylation, suggesting that PTP-MEG2 may be a potential antidiabetic target. In this study, we found that phloridzin, isolated from Ulmus davidiana var. japonica, inhibits the catalytic activity of PTP-MEG2 (half-inhibitory concentration, IC₅₀ = 32 ± 1.06 μM) in vitro, indicating that it could be a potential antidiabetic drug candidate. Importantly, phloridzin stimulated glucose uptake by differentiated 3T3-L1 adipocytes and C2C12 muscle cells compared to that by the control cells. Moreover, phloridzin led to the enhanced phosphorylation of AMPK and Akt relevant to increased insulin sensitivity. Importantly, phloridzin attenuated palmitate-induced insulin resistance in C2C12 muscle cells. We also found that phloridzin did not accelerate adipocyte differentiation, suggesting that phloridzin improves insulin sensitivity without significant lipid accumulation. Taken together, our results demonstrate that phloridzin, an inhibitor of PTP-MEG2, stimulates glucose uptake through the activation of both AMPK and Akt signaling pathways. These results strongly suggest that phloridzin could be used as a potential therapeutic candidate for the treatment of type 2 diabetes.