Role of Perivascular Adipose Tissue in Aorta Reactivity from Obese and Hyperglycemic CD-1 Mice: New Insights into Perivascular Adipose Tissue.

Background: Perivascular adipose tissue (PVAT) plays an essential role in cardiovascular homeostasis. However, during obesity and diabetes, its role in vascular tone regulation is unclear. This study aimed to evaluate the function of the PVAT on aorta reactivity in the lean and cafeteria (CAF) diet-induced obese-hyperglycemic mice model. Methods: Aorta reactivity to phenylephrine, KCl, and acetylcholine was analyzed in lean (n = 6) and obese mice (n = 6). Also, nitric oxide (NO-) and cyclooxygenase participation, in the presence (n = 6) and absence (n = 6) of PVAT, were examined in the aortas. Results: After a CAF diet for 19 weeks, obese mice showed increased body weight, glucose intolerance, and hypercholesterolemia concerning lean mice. Vascular reactivity to phenylephrine was reduced significantly in the aorta of obese mice. In contrast, the contraction produced by KCl (80 mM) was increased in the aorta of obese mice independent of PVAT. Acetylcholine-induced vasorelaxation diminished in the aortas of obese mice in the presence of PVAT. Nonselective inhibition of cyclooxygenases likely shows that PVAT and endothelium release vasorelaxant prostanoids. Conclusions: The results suggest that PVAT modulates aorta reactivity by releasing NO-, decreasing the α1-adrenergic response to phenylephrine, and probably releasing vasorelaxant prostanoids. The data suggest that PVAT regulates the vascular smooth muscle and endothelial function in a CAF diet-induced obese-hyperglycemic mice model.

Antihyperglycemic Effects of Salvia polystachya Cav. and Its Terpenoids: α-Glucosidase and SGLT1 Inhibitors.

The antihyperglycemic activity of ethanolic extract from Salvia polystachya (EESpS) and its products was evaluated using in vivo, ex vivo and in silico assays; additionally, an acute toxicity assay was evaluated. EESpS was classified as a nontoxic class 5 drug. EESpS, ethyl acetate fraction (EtOAcFr), secondary-6-fraction (SeFr6), ursolic acid (UA), and oleanolic acid (OA) reduced the hyperglycemia in DM2 mice. α-glucosidase inhibition was evaluated with oral sucrose and starch tolerance tests (OSuTT and OStTT), an intestinal sucrose hydrolysis (ISH) assay and molecular docking studies using acarbose as control. SGLT1 inhibition was evaluated with oral glucose and galactose tolerance tests (OGTT and OGaTT), an intestinal glucose absorption (IGA) assay and molecular docking studies using canagliflozin as the control. During the carbohydrate tolerance tests, all the treatments reduced the postprandial peak, similar to the control drugs. During the ISH, IC50 values of 739.9 and 726.3 µM for UA and OA, respectively, were calculated. During the IGA, IC50 values of 966.6 and 849.3 for UA, OA respectively, were calculated. Finally, during the molecular docking studies, UA and OA showed ∆G values of -6.41 and -5.48 kcal/mol-1, respectively, on α-glucosidase enzymes. During SGLT1, UA and OA showed ∆G values of -10.55 and -9.65, respectively.

Cannabis and cannabinoids as an alternative remedy in metabolic syndrome

Abstract Metabolic syndrome (MetS), an epidemic defined as a group of interconnected physiological, biochemistry, clinical, and metabolic factors, directly increases the risk of cardiovascular disease, atherosclerosis, type 2 diabetes, and death. MetS therapy includes diet, physical exercise, and a poly-pharmacological intervention. Cannabis is mainly recognized for its recreational uses and has several medical applications for neurological diseases, due to its hypnotic, anxiolytic, antinociceptive, anti-inflammatory, and anticonvulsant activities. Although several clinical observations in Cannabis smokers suggest metabolic effects, its utility in metabolic disorders is unclear. This review aims to determine under what conditions Cannabis might be useful in the treatment of MetS. Cannabis contains 120 phytocannabinoids, of which Δ9-THC mediates its psychoactive effects. Cannabinoids exert biological effects through interactions with the endocannabinoid system, which modulates several physiologic and metabolic pathways through cannabinoid receptors (CB1/CB2). Signaling through both receptors inhibits neurotransmitter release. In general, endocannabinoid system stimulation in Cannabis smokers and Δ9-THC signaling through CB1 have been implicated in MetS development, obesity, and type 2 diabetes. In contrast, CB1 antagonists and non-psychotropic phytocannabinoids like cannabidiol reduce these effects through interactions with both cannabinoid and non-cannabinoid receptors. These pharmacological approaches represent a source of new therapeutic agents for MetS. However, more studies are necessary to support the therapeutic potential of Cannabis and cannabinoids in metabolic abnormalities

GLUT4 translocation in C2C12 myoblasts and primary mouse hepatocytes by an antihyperglycemic flavone from Tillandsia usneoides.

BACKGROUND: Type 2 Diabetes (T2D) is characterized by deregulation in carbohydrate and lipid metabolism, with a very high mortality rate. Glucose Transporter type 4 (GLUT4) plays a crucial role in T2D and represents a therapeutic target of interest. Tillandsia usneoides (T. usneoides) is a plant used as a remedy for diabetes. T. usneoides decreased blood glucose in different experimental models. However, the involvement of GLUT4 in this effect has not yet been explored. PURPOSE: This study aimed to investigate whether any component in T. usneoides might participate in the effect on blood glucose through a bioassay-guided fractionation, testing its potential antihyperglycemic effect in mice, as well as its influence on GLUT4 translocation in C2C12 myoblasts and primary hepatocytes. METHODS: The aqueous extract and the Ethyl Acetate fraction (TU-AcOEt) of T. usneoides were evaluated in a hypoglycemic activity bioassay and in the glucose tolerance test in CD-1 mice. TU-AcOEt was fractionated, obtaining five fractions that were studied in an additional glucose tolerance test. C1F3 was fractioned again, and its fractions (C2F9-12, C2F22-25, and C2F38-44) were examined by HPLC. The C2F38-44 fraction was analyzed by Mass Spectrometry (MS) and subjected to additional fractionation. The fraction C3F6-9 was explored by Nuclear Magnetic Resonance (NMR), resulting in 5,7,4´-trihydroxy-3,6,3´,5´-tetramethoxyflavone (Flav1). Subsequently, a viability test was performed to evaluate the cytotoxic effect of Flav1 and fractions C2F9-12, C2F22-25. C2F38-44, and C3F30-41 in C2C12 myoblasts and primary mouse hepatocytes. Confocal microscopy was also performed to assess the effect of Flav1 and fractions on GLUT4 translocation. RESULTS: The TU-AcOEt fraction exhibited a hypoglycemic and antihyperglycemic effect in mice, and its fractionation resulted in five fractions, among which fraction C1F3 decreased blood glucose. MS and NMR analysis revealed the presence of Flav1. Finally, Flav1 significantly promoted the translocation of GLUT4 in C2C12 myoblasts and primary hepatocytes. CONCLUSION: To date, Flav1 has not been reported to have activity in GLUT4; this study provides evidence that T. usneoides is a plant with the potential to develop novel therapeutic agents for the control of T2D.

Glycine is a competitive antagonist of the TNF receptor mediating the expression of inflammatory cytokines in 3T3-L1 adipocytes.

OBJECTIVE: To determine the involvement of TNF-α and glycine receptors in the inhibition of pro-inflammatory adipokines in 3T3-L1 cells. METHODS: RT-PCR evidenced glycine receptors in 3T3-L1 adipocytes. 3T3-L1 cells were transfected with siRNA for the glycine (Glrb) and TNF1a (Tnfrsf1a) receptors and confirmed by confocal microscopy. Transfected cells were treated with glycine (10 mM). The expressions of TNF-α and IL-6 mRNA were measured by qRT-PCR, while concentrations were quantified by ELISA. RESULTS: Glycine decreased the expression and concentration of TNF-α and IL-6; this effect did not occur in the absence of TNF-α receptor due to siRNA. In contrast, glycine produced only slight changes in the expression of TNF-α and IL-6 in the absence of the glycine receptor due to siRNA. A docking analysis confirmed the possibility of binding glycine to the TNF-α1a receptor. CONCLUSION: These findings support the idea that glycine could partially inhibit the binding of TNF-α to its receptor and provide clues about the mechanisms by which glycine inhibits the secretion of pro-inflammatory adipokines in adipocytes through the TNF-α receptor.

Jatropha Neopauciflora Pax Latex Exhibits Wound-Healing Effect in Normal and Diabetic Mice.

Jatropha neopauciflora is an endemic species of Mexico. Its latex is used to treat wounds, scarring, oral infections, and loose teeth. To date, there are no studies that validate at a morphological level a wound-healing use in diabetes. The present research aimed to evaluate the wound-healing capacity of the latex of J. neopauciflora in the skin of healthy and streptozotocin-induced diabetic mice. Also, a chemical analysis of the latex through molecular exclusion chromatography and HPLC were performed. Male mice (Mus musculus) of 7-week-old CD1 strain were used. Groups of healthy and diabetic mice were formed. A longitudinal cut of 1 cm was performed on the depilated skin. All treatments were topically applied to the wound area twice a day for ten days. At the end of the experiments, the skin sections were obtained from the wound area and stained with Hematoxylin-Eosin. Then we counted the number of active fibroblasts in all the experimental groups. In normal mice, the latex accelerated the wound-healing process and decreased the number of active fibroblasts, similarly to Recoveron. In diabetic mice, the latex and Recoveron increased the number of active fibroblasts. In normal and diabetic mice, a thin and orderly epidermis was observed. Molecular exclusion chromatography exhibited 58 fractions, 14 of which were subjected to HPLC, to detect catechin, a flavonoid with antioxidant, antimicrobial, and anti-inflammatory properties. J. neopauciflora latex can be useful for wound treatment in patients with diabetes mellitus because it accelerates and promotes the wound-healing process.

α-Amyrin induces GLUT4 translocation mediated by AMPK and PPARδ/γ in C2C12 myoblasts.

α-Amyrin, a natural pentacyclic triterpene, has an antihyperglycemic effect in mice and dual PPARδ/γ action in 3T3-L1 adipocytes, and potential in the control of type 2 diabetes (T2D). About 80% of glucose uptake occurs in skeletal muscle cells, playing a significant role in insulin resistance (IR) and T2D. Peroxisome-proliferator activated receptors (PPARs), in particular PPARδ and PPARγ, are involved in the regulation of lipids and carbohydrates and, along with adenosine-monophosphate (AMP) - activated protein kinase (AMPK) and protein kinase B (Akt), are implicated in translocation of glucose transporter 4 (GLUT4); however, it is still unknown whether α-amyrin can affect these pathways in skeletal muscle cells. Our objective was to determine the action of α-amyrin in PPARδ, PPARγ, AMPK, and Akt in C2C12 myoblasts. The expression of PPARδ, PPARγ, fatty acid transporter protein (FATP), and GLUT4 was quantified using reverse transcription quantitative PCR and Western blot. α-Amyrin increased these markers along with phospho-AMPK (p-AMPK) but not p-Akt. Molecular docking showed that α-amyrin acts as an AMPK-allosteric activator, and may be related to GLUT4 translocation, as evidenced by confocal microscopy. These data support that α-amyrin could have an insulin-mimetic action in C2C12 myoblasts and should be considered as a bioactive molecule for new multitarget drugs with utility in T2D and other metabolic diseases.

Oleanolic acid induces a dual agonist action on PPARγ/α and GLUT4 translocation: A pentacyclic triterpene for dyslipidemia and type 2 diabetes.

Type 2 diabetes (T2D) is a metabolic disease characterized by defects in glycemia regulation. This disease is associated with alterations in insulin action and lipid metabolism, generating hyperglycemia and dyslipidemias. Currently, it is necessary to develop new or known drugs that promote the sensitization of insulin action. Thus, activation of peroxisome proliferator-activated receptors (PPARs) is probably the key to doing this. PPARs participate in maintaining an energetic balance between storage and the expenditure of energy. The activation of PPARγ produces the storage of energy, mainly as glycogen and fat. Meanwhile, PPARα activation promotes lipid degradation. Oleanolic acid (OA), a pentacyclic triterpenoid of numerous edible and medicinal plants, decreases hyperglycemia and lipid accumulation. However, the effects on PPARs and their regulated genes are unknown. Our aim was to determine the effects of OA on PPAR γ/α expression and their regulated genes (adiponectin, type 4 glucose transporter, fatty acid transport protein, and long-chain acyl-CoA synthetase) in C2C12 myoblasts by RT-PCR, Western blot, GLUT-4 translocation, and lipid storage in 3T3-L1 adipocytes. In C2C12 myoblasts, OA increased the expression of mRNA in both PPARγ/α and their regulated genes; also, PPARγ, GLUT-4, and FATP-1 protein expression increased, as well as GLUT-4 translocation. In 3T3-L1, OA increased the expression of mRNA in both PPARγ/α and maintained lipid storage unchanged. In conclusion, OA exhibited a dual action on PPARγ/α, which might explain in part its antihyperglycemic effect. This compound represents an alternative for designing novel therapeutic strategies in the control of T2D.

Mechanism of the Hypoglycemic Activity and Hepatoprotective Effect of the Aqueous Extract of Cecropia obtusifolia Bertol.

In Central and South American traditional medicine, people use Cecropia obtusifolia Bertol (Cecropiaceae) for the treatment of diabetes mellitus. However, its hypoglycemic action mechanism at pancreatic and liver level has been poorly explored. The present research aimed to establish the influence of the aqueous extract of C. obtusifolia, standardized in its content of chlorogenic acid, on insulin secretion in RINm5F cells and over the liver carbohydrates and lipids metabolism, and to determine concomitantly its hepatoprotective effect on mice with streptozotocin-induced diabetes. In RINm5F cells, concentrations 5, 50, 100, and 200 µg/mL of aqueous extract of C. obtusifolia were used to determine [Ca2+]i and insulin secretion. In an acute study, the extract was administered at doses of 500 mg/kg. In another test (subacute), the extract was daily administrated to diabetic mice (200 mg/kg/day) for 30 days. Blood glucose levels and other biochemical parameters were determined, and a liver histological analysis was performed. In RINm5F cells, C. obtusifolia increased [Ca2+]i and insulin secretion, whereas in diabetic mice exhibited acute and subacute hypoglycemic effects. Daily administration of C. obtusifolia to diabetic mice also increased liver glycogen storage and glycogen synthase levels, without apparent changes in gluconeogenesis. Besides, it increased peroxisome proliferator-activated receptor-α (PPAR-α) and long-chain-fatty-acid-CoA ligase 1 (ACSL-1) expression and reduced triglycerides, transaminases (alanine aminotransferase and aspartate aminotransferase), and collagen fibers, modifying anti-inflammatory (adiponectin and interleukin-10) and inflammatory (tumor necrosis factor-α) cytokines in serum. Therefore, the hypoglycemic effect of C. obtusifolia implicates a dual action, promoting insulin secretion, liver glycogen accumulation, and hepatoprotection by decreasing collagen fibers and inflammatory markers, whereas it improves lipid metabolism, due in part to PPAR-α.

Ayahuasca: Uses, Phytochemical and Biological Activities.

Ayahuasca (caapi, yajé), is a psychoactive brew from the Amazon Basin region of South America traditionally considered a "master plant." It is prepared as a decoction from Banisteriopsis caapi and Psychotria viridis, which it is thought that it stimulates creative thinking and visual creativity. Native healers of the Orinoco and Amazon basins have used traditionally ayahuasca as a healing tool for multiple purposes, particularly to treat psychological disorders in the patients, with some beneficial effects experimentally and clinically validated. Recently, several syncretic religions, as the "União de Vegetal" (UDV) group in Brazil, have been spread around the world. The use of ayahuasca has been popularized by internet and smart-shops, bringing the psychoactive substance to new highs, emerging new "ayahuasqueros." Ayahuasca has alkaloids as ß-carbolines and dimethyltryptamines, which inhibit the monoamine oxidase and active the 5-HT2A (5-hydroxytryptamine) receptor, respectively, resulting in hallucinations in the users. Ayahuasca induces a psychedelic change in the anteroposterior coupling of the electrophysiological brain oscillations in humans. Traditional ayahuasca beverage is generating pharmacological, commercial and spiritual interest among the scientific community, government people, and different populations worldwide. The goal of this article is to report about the uses, chemistry and biological activities of ayahuasca.

The Tillandsia genus: history, uses, chemistry, and biological activity / El género Tillandsia: historia, usos, química y actividad biológica

Tillandsia L. genus comprises 649 species, with different uses at different times. T. usneoides L. uses are reported since the late- archaic and pre-Columbian cultures. In XIX-XX centuries, T. usneoides was used in some manufactured products, as polish and packing fruit. Tillandsia has a favorable reputation as medicine: for leucorrhea, rheumatism, ulcers, hemorrhoid treatment, as an anti-diabetic remedy, emetic, analgesic, purgative, contraceptive, antispasmodic and diuretic. Tillandsia chemical composition includes cycloartane triterpenes and hydroxy-flavonoids, which are present in at least 24 species. Several extracts and compounds from Tillandsia spp. have been reported with pharmacological actions, as anti-neoplasia, hypolipidemic, antifungal, anti-HSV-1, hypoglycemic and microbicide. This review communicates the economic importance, ethnobotany, chemistry composition and biological activities of the Tillandsia genus, and analyze its biological and economic perspective. Tillandsia genus has cultural, economic and pharmacological relevance, with a high potential in many essential aspects of the modern society.

El género Tillandsia L. comprende 649 especies, con diferentes usos en diferentes épocas. T. usneoides L. se han reportado desde el arcáico tardío hasta las culturas precolombinas. En los siglos XIX-XX, T. usneoides se usó en productos manufacturados: como abrasivo y embalaje de fruta. Como medicina tradicional, el género Tillandsia se reporta para leucorrea, reumatismo, úlceras, hemorroides, remedio antidiabético, emético, analgésico, purgante, anticonceptivo, antiespasmódico y diurético. Su composición química incluye triterpenos de tipo ciclo-artano e hidroxi-flavonoides, presentes en al menos 24 especies. Los extractos y compuestos del género Tillandsia se han reportado con propiedades antineoplásicas, hipolipidémicas, antifúngicas, anti-HSV-1, hipoglucemiantes y microbicidas. Esta revisión comunica la importancia económica, etnobotánica, composición química y las actividades biológicas del género Tillandsia, y analiza su perspectiva biológica y potencial económica. Tillandsia tiene importancia cultural, económica y farmacológica, con gran potencial en muchos aspectos esenciales de la sociedad moderna.

Triterpenoids from Hibiscus sabdariffa L. with PPARδ/γ Dual Agonist Action: In Vivo, In Vitro and In Silico Studies.

Hibiscus sabdariffa is a medicinal plant consumed as a diuretic and anti-obesity remedy. Several pharmacological studies have shown its beneficial effects in metabolism. Peroxisome proliferator-activated receptors δ and γ may play a role in the actions of H. sabdariffa. These nuclear receptors regulate lipid and glucose metabolism and are therapeutic targets for type 2 diabetes. This research aimed to perform a phytochemical study guided by a bioassay from H. sabdariffa to identify compounds with peroxisome proliferator-activated receptor δ and peroxisome proliferator-activated receptor γ agonist activity, supported by messenger ribonucleic acid expression, molecular docking, lipid accumulation, and an antihyperglycemic effect. An oral glucose tolerance test in mice with the aqueous extract of H. sabdariffa and the dichloromethane extract of H. sabdariffa was performed. The dichloromethane extract of H. sabdariffa exhibited an antihyperglycemic effect. The dichloromethane extract of H. sabdariffa was fractioned, and four fractions were evaluated in 3T3-L1 adipocytes on peroxisome proliferator-activated receptor δ, peroxisome proliferator-activated receptor γ, fatty acid transporter protein, and glucose transporter type 4 messenger ribonucleic acid expression. Fraction F3 exhibited peroxisome proliferator-activated receptor δ/γ dual agonist activity, and a further fractionation yielded two subfractions, F3-1 and F3-2, which also increased peroxisome proliferator-activated receptor δ and peroxisome proliferator-activated receptor γ expression. Subfractions were analyzed by GC/MS. The main compounds identified in F3-1 were linoleic acid, oleic acid, and palmitic acid, while in F3-2, the main compounds identified were α-amyrin and lupeol. These molecules were subjected to molecular docking analysis. α-Amyrin and lupeol showed the highest affinity. Moreover, both produced an increase in peroxisome proliferator-activated receptor δ, peroxisome proliferator-activated receptor γ, fatty acid transporter protein, and glucose transporter type 4 expression. Additionally, α-amyrin and lupeol decreased lipid accumulation in 3T3-L1 adipocytes and blood glucose in mice. Until now, α-amyrin and lupeol have not been reported with activity on peroxisome proliferator-activated receptors. This study provides evidence that α-amyrin and lupeol possess antidiabetic effects through a peroxisome proliferator-activated receptor δ/γ dual agonist action.

A Phenolic Fraction from Catharanthus roseus L. Stems Decreases Glycemia and Stimulates Insulin Secretion.

Catharanthus roseus (L.) G. (C. roseus) is a medicinal plant used traditionally for diabetes mellitus control. Several compounds of an alkaloidal nature have been proposed as hypoglycemic principles. However, little attention has been paid to other compounds in this plant that could also participate in this hypoglycemic activity. This study aimed to analyze the hypoglycemic effect of a polyphenolic fraction from C. roseus, as well as its action on insulin secretion and expression in RINm5F cells. Methods. An alkaloid-free aqueous extract was obtained from C. roseus stems. The hypoglycemic effect of different doses of this extract was evaluated in normal and streptozotocin-induced diabetic mice. This extract was fractionated by bipartition, and the resultant fractions were assessed by their hypoglycemic effects. Subsequently, the fraction with the greater hypoglycemic activity was added to the RINm5F cells, and the expression and secretion of insulin were analyzed. The antioxidant activity was determined by the DPPH method and through chromatographic analysis of the most active fraction by HPLC, using an Econosphere C18 column. Results. The aqueous alkaloid-free extract of C. roseus stems significantly reduced blood glucose in normal and diabetic mice. The fractionation of this extract provided three fractions, one of which (a precipitate) showed significant reductions in glycemia at 6 h (48.1 and 64.5% in normal and diabetic mice, respectively). This precipitate contained phenolic compounds and saponins. Its chromatographic analysis showed that it is formed by several phenolic compounds; gallic acid (0.053%) and chlorogenic acid (0.216%) were identified and quantified. Conclusion. The phenolic fraction of C. roseus containing gallic acid and chlorogenic acid had a hypoglycemic effect that may be explained by an increase in insulin secretion.

Participation of the IKK-α/ß complex in the inhibition of the TNF-α/NF-κB pathway by glycine: Possible involvement of a membrane receptor specific to adipocytes.

Glycine modulates inflammatory processes mediated by macrophages and adipocytes through decreasing the secretion of TNF-α, IL-6, and leptin, while increasing adiponectin. These effects have been associated with the inactivation of NF-κB in response to TNF-α, across an increase of its inhibitor IκB-α in adipocytes. However, glycine upstream mainly influences the IκB kinase (IKK) complex, a multi-protein kinase complex considered a critical point in regulation of the NF-κB pathway; whether that is responsible for the TNF-α-induced phosphorylation of IkB has not been explored. Additionally, although previous studies have described glycine interactions with specific receptors (GlyR) in different immune system cell types, it is currently unknown whether adipocytes present GlyR. In this research, participation of the IKK-α/ß complex in the inhibition of the TNF-α/NF-κB pathway by glycine was evaluated and associated with the synthesis and secretion of inflammatory cytokines in 3T3-L1 adipocytes. Furthermore, we also explored GlyR expression, its localization on the plasmatic membrane, intracellular calcium concentrations [Ca2+]i and strychnine antagonist action over the GlyR in these cells. Glycine decreased the IKK-α/ß complex and the phosphorylation of NF-κB, diminishing the expression and secretion of IL-6 and TNF-α, but increasing that of adiponectin. GlyR expression and its fluorescence in the plasma membrane were increased in the presence of glycine. In addition, glycine decreased [Ca2+]i; whereas strychnine + glycine treatment inhibited the activation of NF-κB observed with glycine. In conclusion, the reduction of TNF-α and IL-6 and suppression of the TNF-α/NF-κB pathway by glycine may be explained in part by inhibition of the IKK-α/ß complex, with a possible participation of GlyR in 3T3-L1 adipocytes.

Potential of the chlorogenic acid as multitarget agent: Insulin-secretagogue and PPAR α/γ dual agonist.

The chlorogenic acid (CGA) is a natural product isolated from Cecropia obtusifolia, which possesses several pharmacological properties, such as: anti-carcinogenic, neuroprotective, antioxidant, anti-inflammatory, hypoglycemic, and hypolipidemic. In relation to its effects on the hyperglycemia and hypertriglyceridemia, few is known about the mechanisms in which this compound may be acting, therefore, the aim of the present study was to determine if CGA acts as an insulin secretagogue increasing intracellular calcium concentrations ([Ca2+]i) in RINm5F cells; or as an insulin sensitizer and lipid-lowering agent stimulating the expression of PPARγ and PPARα, respectively, in 3T3-L1 adipocytes. As results, RINm5F cells treated with 200µM of CGA showed an increase in [Ca2+]i of 9-times versus control and 4-times as compared to positive control; in addition, an increase in insulin secretion was observed similarly to those of positive control. CGA also significantly increased the mRNA expression of PPARγ (150%) and GLUT4 (220%), as well PPARα (40%) and FATP (25%) as it was appreciated by RT-PCR. Additionally, a chemoinformatic analysis suggested that CGA has suitable physicochemical properties to be considered as leader bioactive molecule for the development of novel agents with similar properties. Together, our results indicate that CGA possesses multiple mechanisms of action for the development of highly effective therapeutics in the treatment of metabolic diseases such as type 2 diabetes.

CHEMICAL CHARACTERIZATION OF A HYPOGLYCEMIC EXTRACT FROM CUCURBITA FICIFOLIA BOUCHE THAT INDUCES LIVER GLYCOGEN ACCUMULATION IN DIABETIC MICE.

BACKGROUND: The aqueous extract of Cucurbita ficifolia (C. ficifolia) fruit has demonstrated hypoglycemic effect, which may be attributed to some components in the extract. However, the major secondary metabolites in this fruit have not yet been identified and little is known about its extra-pancreatic action, in particular, on liver carbohydrate metabolism. Therefore, in addition to the isolation and structural elucidation of the principal components in the aqueous extract of C. ficifolia, the aim of this study was to determine whether or not the hypoglycemic effect of the aqueous extract of Cucurbita ficifolia (C. ficifolia) fruit is due to accumulation of liver glycogen in diabetic mice. MATERIALS AND METHODS: The aqueous extract from fruit of C. ficifolia was fractionated and its main secondary metabolites were purified and chemically characterized (NMR and GC-MS). Alloxan-induced diabetic mice received daily by gavage the aqueous extract (30 days). The liver glycogen content was quantified by spectroscopic method and by PAS stain; ALT and AST by spectrometric method; glycogen synthase, glycogen phosphorylase and GLUT2 by Western blot; the mRNA expression of GLUT2 and glucagon-receptor by RT-PCR; while serum insulin was quantified by ELISA method. A liver histological analysis was also performed by H&E stain. RESULTS: Chemical fingerprint showed five majoritarian compounds in the aqueous extract of C. ficifolia: p-coumaric acid, p-hydroxybenzoic acid, salicin, stigmast-7,2,2-dien-3-ol and stigmast-7-en-3-ol. The histological analysis showed accumulation of liver glycogen. Also, increased glycogen synthase and decreased glycogen phosphorylase were observed. Interestingly, the histological architecture evidenced a liver-protective effect due the extract. CONCLUSION: Five compounds were identified in C. ficifolia aqueous extract. The hypoglycemic effect of this extract may be partially explained by liver glycogen accumulation. The bioactive compound responsible for the hypoglycemic effect of this extract will be elucidated in subsequent studies.

Discovery of new dual PPARγ-GPR40 agonists with robust antidiabetic activity: Design, synthesis and in combo drug evaluation.

The design of compounds 1 and 2 was based on the similar scaffold of pharmacophoric groups for PPARγ and GPR40 agonists. In order to find new compounds with improved biological activity, the current manuscript describes a new dual PPARγ-GPR40 agonist. We synthesized two compounds, which were prepared following a multistep synthetic route, and the relative mRNA expression levels of PPARγ, GLUT4, and GPR40 were quantified in cell culture, as well as insulin secretion and [Ca2+] intracellular levels. Compound 1 showed a 7-times increase in the mRNA expression of PPARγ, which in turn enhanced the expression levels of GLUT4 respect to control and pioglitazone. It also showed an increase of 2-fold in the [Ca2+]i level allowing an increment on insulin release, being as active as the positive control (glibenclamide), causing also an increase of 2-fold in mRNA expression of GPR40. Furthermore, the compound 2 showed lower activity than the compound 1. The ester of 1 showed antidiabetic activity at a 50mg/kg single dose in streptozotocin-nicotinamide-induced diabetic mice model. In addition, we achieved a molecular docking study of compound 1 on PPARγ and GPR40 receptors, showing a great affinity for both targets. We observed important polar interactions between the carboxylic group and main residues into the binding pocket. Therefore, the compound 1 has a potential for the development of antidiabetic agents with newfangled dual action.

Fermented blueberry juice extract and its specific fractions have an anti-adipogenic effect in 3 T3-L1 cells.

BACKGROUND: Obesity and Type 2 diabetes have reached epidemic status worldwide. Wild lowbush blueberry (Vaccinium angustifolium Aiton) is a plant of the North American Aboriginal traditional pharmacopeia with antidiabetic potential, especially when it is fermented with Serratia vaccinii. METHODS: A phytochemical fractionation scheme was used to identify potential bioactive compounds as confirmed by HPLC retention times and UV-Vis spectra. 3 T3-L1 cells were differentiated for 7 days with either Normal Blueberry Extract (NBE), Fermented Blueberry Extract (FBE/F1), seven fractions and four pure compounds. Triglyceride content was measured. Examination of selected intracellular signalling components (p-Akt, p-AMPK) and transcriptional factors (SREBP-1c and PPARγ) was carried out by Western blot analysis. RESULTS: The inhibitory effect of FBE/F1 on adipocyte triglyceride accumulation was attributed to total phenolic (F2) and chlorogenic acid enriched (F3-2) fractions that both inhibited by 75%. Pure compounds catechol (CAT) and chlorogenic acid (CA) also inhibited adipogenesis by 70%. Treatment with NBE, F1, F3-2, CAT and CA decreased p-AKT, whereas p-AMPK tended to increase with F1. The expression of SREBP1-c was not significantly modulated. In contrast, PPARγ decreased in all experimental groups that inhibited adipogenesis. CONCLUSIONS: These results demonstrate that fermented blueberry extract contains compounds with anti-adipogenic activity, which can serve to standardize nutraceutical preparations from fermented blueberry juice and to develop novel compounds with anti-obesity properties.

Antidiabetic, antihyperlipidemic and anti-inflammatory effects of tilianin in streptozotocin-nicotinamide diabetic rats.

Flavonoids from medicinal plants have been used in traditional medicine to treat a variety of prevalent diseases. Flavones activate the signaling pathways promoting fuel metabolism and insulin sensitizing in hepatocytes and adipocytes, which suggests that flavones may have the potential to exert in vivo antidiabetic and antihyperlipidemic effects. Thus, the aim of the current study was to determine the antidiabetic, antihyperlipidemic and anti-inflammatory effects of tilianin in diabetic rats. Also, to understand the mechanism involved using in vitro 3T3-L1 cells and tissues from experimental animals treated with test samples through molecular profile studies. Non insulin-dependent diabetic mellitus (NIDDM) rats were treated over a short period (for 10 days) with 60mg/Kg/day of tilianin. After treatment, a biochemical blood profile was determined. Also, adipose and thoracic aortic tissues were used to determine pro-inflammatory profile, adiponectin and adhesion molecules by real-time PCR. In 3T3-L1 cells pretreated with tilianin (10µM), PPARα, PPARγ, GLUT4, FATP-1 and ACSL-1 mRNA expression were measured. In order to explain the potential PPARα interaction with tilianin, a docking study with PPARα was carried out. Thus, intragastric administration of tilianin and metformin induced a decrease in plasma glucose (GLU) in diabetic rats on day 6, and remained significantly lower until the end of the treatment; also blood triacylglycerides (TAG) and cholesterol (CHOL) (p<0.05) were diminished. Moreover, IL-1ß and IL-18 expression was significantly decreased in adipose tissue (p<0.05); meanwhile adiponectin was significantly overexpressed (p<0.05). Besides, ICAM-1 expression was significantly reduced in aortic tissue (p<0.05). In 3T3-L1 cells it was found that tilianin increased PPARα and ACSL1 mRNA levels (p<0.05). Finally, tilianin docking studies with PPARα showed polar interactions with Glu269, Tyr314, His 440 and Tyr464 residues. In conclusion, short-term tilianin treatment might exert its antidiabetic and antihyperlipidemic effect by modulating a pro-inflammatory profile, and increasing adiponectin expression. In addition, our results suggest the possible interaction of tilianin with PPARα.

Cucurbita ficifolia Bouché increases insulin secretion in RINm5F cells through an influx of Ca(2+) from the endoplasmic reticulum.

ETHNOPHARMACOLOGICAL IMPORTANCE: Cucurbita ficifolia Bouché(C. ficifolia) is a plant used in Mexican traditional medicine to control type 2 diabetes (T2D). The hypoglycemic effect of the fruit of C. ficifolia has been demonstrated in different experimental models and in T2D patients. It has been proposed that D-chiro-inositol (DCI) is the active compound of the fruit. Additionally, it has been reported that C. ficifolia increases the mRNA expression of insulin and Kir 6.2 (a component of the ATP-sensitive potassium (K(+)ATP) channel, which is activated by sulphonylurea) in RINm5F cells. However, it remains unclear whether C. ficifolia and DCI causes the secretion of insulin by increasing the concentration of intracellular calcium ([Ca(2+)]i) through K(+)ATP channel blockage or from the reservoir in the endoplasmic reticulum (ER). MATERIAL AND METHODS: The aqueous extract of C. ficifolia was obtained and standardized with regard to its DCI content. RINm5F pancreatic ß-cells were incubated with different concentrations (50, 100, 200 and 400µM) of DCI alone or C. ficifolia (9, 18, 36 and 72µg of extract/mL), and the [Ca(2+)]i of the cells was quantified. The cells were preloaded with the Ca(2+) fluorescent dye fluo4-acetoxymethyl ester (AM) and visualized by confocal microscopy. Insulin secretion was measured by an ELISA method. Subsequently, the effect of C. ficifolia on the K(+)ATP channel was evaluated. In this case, the blocker activator diazoxide was used to inhibit the C. ficifolia-induced calcium influx. In addition, the inositol 1,4,5-trisphosphate (IP3)-receptor-selective inhibitor 2-amino-thoxydiphenylborate (2-APB) was used to inhibit the influx of calcium from the ER that was induced by C. ficifolia. RESULTS: It was found that DCI alone did not increase [Ca(2+)]i or insulin secretion. In contrast, treatment with C. ficifolia increased [Ca(2+)]i 10-fold compared with the control group. Insulin secretion increased by 46.9%. In the presence of diazoxide, C. ficifolia decreased [Ca(2+)]i by 50%, while insulin secretion increased by 36.4%. In contrast, in the presence of 2-APB, C. ficifolia increased [Ca(2+)]i 18-fold, while insulin secretion remained constant, indicating an additive effect. Therefore, C. ficifolia was not found to block the K(+)ATP channel. However, it did exert an effect by increasing [Ca(2+)]i from the ER, which may partly explain the insulin secretion observed following treatment with C. ficifolia. CONCLUSIONS: The hypoglycemic properties of C. ficifolia can be explained in part by its effect as a secretagogue for insulin through an increase in [Ca(2+)]i from the calcium reservoir in the ER. Therefore, the mechanism of action of C. ficifolia is different to those of the currently used hypoglycemic drugs, such as sulfonylureas. These results support that C. ficifolia may be a potential natural resource for new agents to control T2D.