Inhibitory effect of hypocrellin A on protein kinase C in liver and skeletal muscle of obese Zucker rats

In this ex vivo study, the inhibitory activity of hypocrellin A (HA), a perylene quinonoid pigment isolated from the Chinese medicinal fungus Hypocrella bambuase, on protein kinase C (PKC) enzyme activity in insulin target tissues of obese Zucker rats was assessed. Pre-incubation with HA for 30 minutes significantly inhibited the activity of partially purified PKC enzyme from liver and soleus skeletal muscle in a dose-dependent manner (IC50 = 0.07 and 0.26 microg/ml, respectively). HA produced a greater inhibitory effect in enzyme prepared from the liver than enzyme prepared from soleus muscle. Since total PKC activity in these two insulin target tissues is the net result of several different isoforms of PKC, and PKC-theta is a major isoform expressed in the soleus skeletal muscle, the present data suggest that the naturally occurring compound, HA, may selectively inhibit certain PKC isoforms other than PKC-theta. Further investigations are required to determine which PKC isoforms are most susceptible to HA and whether changes in PKC signaling during treatment with HA can reverse abnormalities of glucose and lipid metabolism in insulin resistant and diabetic states.     

Peroxisome proliferator-activated receptor gamma transactivation-mediated potentiation of glucose uptake by Bai-Hu-Tang

ETHNOPHARMACOLOGICAL RELEVANCE: The molecular mechanism of a traditional hypoglycemic Chinese herbal formula. AIM OF THE STUDY: To explore the glucose uptake effect as well as the mechanism(s) of action of Bai-Hu-Tang (BHT), a traditional Chinese herbal formula, composed of anemarrhena, gypsum, licorice and rice. MATERIALS AND METHODS: Differentiated 3T3-L1 adipocytes were treated with vehicle, insulin or insulin plus different concentrations of BHT. The 2-deoxy-[(3)H] glucose uptake assay was performed to measure the amount of glucose uptake. To explore the mechanism(s) of glucose uptake of BHT, we used two insulin signaling transduction inhibitors, N-Acetyl-Leu-Leu-Norleu-al (ALLN), a calpain inhibitor, and LY 294002, a phosphatidylinositol (PI)3-kinase inhibitor, to test if the glucose uptake effect was mediated by the insulin signaling pathway. We then used Western blot analysis to re-confirm the result of the insulin signaling inhibition assay. Finally, reporter chimera assay of HuH-7 cells was used to measure the peroxisome proliferator-activated receptor gamma (PPARgamma) activation by BHT. RESULTS: BHT potentiated insulin-stimulated glucose uptake in 3T3-L1 adipocytes. The effect of BHT-stimulated glucose uptake was neither inhibited by ALLN nor by LY 294002. The protein expression of PI3 kinase pathway did not change after BHT stimulation. PPARgamma activation was elevated by 67.7+/-32% (p<0.05) in HuH-7 cells treated with 0.8 microg/ml of BHT. CONCLUSIONS: BHT stimulated glucose uptake in 3T3-L1 adipocytes. This effect was via PPARgamma activation rather than via the insulin signaling pathway.     

Amelioration of insulin resistance using the additive effect of ferulic acid and resveratrol on vesicle trafficking for skeletal muscle glucose metabolism

Dysregulation of vesicle trafficking in muscle is one of the factors responsible for the pathogenesis of insulin resistance (IR). Ferulic acid (FER) and resveratrol (RSV) are known to have hypoglycemic property. In this study, differentiated L6 myotubes were induced with palmitate as a model of IR. Chemical ablation of muscle vesicles was used to investigate how FER and RSV influence glucose utilization. Results showed that both FER and RSV elicit glucose uptake and promote glycogen synthesis in insulin‐resistant muscle cells. Mechanistic studies further showed that FER markedly enhances the transferrin receptor‐containing endosomal compartment activities via phosphoinositide 3‐kinase (PI3K)/atypical protein kinase C‐dependent pathway, while RSV facilitates glucose transporter storage vesicles (GSV) trafficking via an exercise‐like effect of conventional protein kinase C/5′‐adenosine monophosphate‐activated protein kinase (AMPK) modulation. Therefore, these two phenolic compounds promoted glucose transport through two separate routes, and they had an additive effect on the increase of glucose uptake in insulin‐resistant muscle cells. These findings provide a basis for the understanding of the antidiabetic potential of RSV and FER combination.     

Beneficial effects of oleuropein on glucose uptake and on parameters relevant to the normal homeostatic mechanisms of glucose regulation in rat skeletal muscle

Oleuropein, the main constituents of leaves and fruits of the olive tree, has been demonstrated to exert various therapeutic and pharmacological properties including antidiabetic effect. However, the effectiveness of oleuropein on glucose homeostasis in intact rat skeletal muscle ex vivo has never been explored. Therefore, our current study was carried out to investigate and confirm the beneficial effect of oleuropein (1.5 mM) on glucose uptake and on parameters relevant to the normal homeostatic mechanisms of glucose regulation in rat skeletal muscle. For this purpose, soleus muscles were incubated for 12 hr without (control) or with oleuropein, in the presence or absence of AMP‐activated protein kinase (AMPK) inhibitor, compound C, or wortmannin, an inhibitor of phosphatidylinositol kinase. Oleuropein‐stimulated glucose transport, plasmalemmal glucose transporter 4 (GLUT4), and phosphorylation of phosphatidylinositol kinase and AMPK were examined. We observed that oleuropein treatment enhanced glucose transport, GLUT4 translocation, and AMPK phosphorylation. The oleuropein‐stimulated glucose uptake and GLUT4 translocation were inhibited by compound C and were not affected by wortmannin. These results suggest that increased glucose uptake induced by oleuropein might be mediated through activation of AMPK and the subsequent increase in GLUT4 translocation in skeletal muscles.     

SDF7, a group of Scoparia dulcis Linn. derived flavonoid compounds,stimulates glucose uptake and regulates adipocytokines in 3T3-F442a adipocytes

Ethnopharmacology relevance

Adipocytes are major tissues involved in glucose uptake second to skeletal muscle and act as the main adipocytokines mediator that regulates glucose uptake mechanism and cellular differentiation. The objective of this study were to examine the effect of the SDF7, which is a fraction consists of four flavonoid compounds (quercetin: p-coumaric acid: luteolin: apigenin=8: 26: 1: 3) from Scoparia dulcis Linn., on stimulating the downstream components of insulin signalling and the adipocytokines expression on different cellular fractions of 3T3-F442a adipocytes.

Material and methods

Morphology and lipid accumulation of differentiated 3T3-F442a adipocytes by 100 nM insulin treated with different concentrations of SDF7 and rosiglitazone were examined followed by the evaluation of glucose uptake activity expressions of insulin signalling downstream components (IRS-1, PI3-kinase, PKB, PKC, TC10 and GLUT4) from four cellular fractions (plasma membrane, cytosol, high density microsome and low density microsome). Next, the expression level of adipocytokines (TNF-α, adiponectin and leptin) and immunoblotting of treated 3T3-F442 adipocytes was determined at 30 min and 480 min. Glucose transporter 4 (GLUT4) translocation of 3T3-F442a adipocytes membrane was also determined. Lastly, mRNA expression of adiponectin and PPAR-γ of 3T3-F442a adipocytes were induced and compared with basal concentration.

Results

It was found that SDF7 was able to induce adipocytes differentiation with great extends of morphological changes, lipid synthesis and lipid stimulation in vitro. SDF7 stimulation of glucose transport on 3T3-F442a adipocytes are found to be dose independent, time-dependent and plasma membrane GLUT4 expression-dependent. Moreover, SDF7 are observed to be able to suppress TNF-α and leptin expressions that were mediated by 3T3-F442a adipocytes, while stimulated adiponectin secretion on the cells. There was a significant expression (p<0.01) of protein kinase C and small G protein TC10 on 3T3-F442a adipocytes upon treatment with SDF7 as compared to the control. SDF7 was also found to be effective in stimulating adiponectin and PPAR-γ mRNA upregulation at 50 µg/ml.

Conclusion

SDF7 exhibited good lipogenesis, adiponectinesis and glucose uptake stimulatory properties on 3T3-F442a adipocytes.     

Coriolus versicolor aqueous extract ameliorates insulin resistance with PI3K/Akt and p38 MAPK signaling pathways involved in diabetic skeletal muscle

Patients with type 2 diabetes mellitus (T2DM) are usually with poor immunity and easier to suffer from cancer and microbial infections. Herein, we report an efficient anti‐diabetic medicinal mushroom, Coriolus versicolor (CV). This study aimed to investigate the anti‐diabetic and anti‐insulin‐resistance effects of CV aqueous extract in myoblasts (L6 cells) and skeletal muscle of T2DM rat. Our results showed that CV extract treatment significantly reduced blood glucose levels of T2DM rats, whereas CV extract increased glucose consumption in insulin resistant L6 cells. Besides, the translocation and expression of glucose transporter 4 were enhanced by CV extract, which indicated that CV extract was effective in diabetic skeletal muscle. Moreover, CV extract treatments resulted in remarkable anti‐insulin‐resistance effects, which was reflected by the change of gene and protein expression levels in PI3K/Akt and p38 MAPK pathways. PI3K inhibitor, LY29004, and p38 MAPK inhibitor, SB203580 confirmed it further. In conclusion, our results demonstrated that the CV extract exhibited anti‐diabetic and anti‐insulin‐resistance effects in diabetic skeletal muscle, and the effects were mediated by PI3K/Akt and p38 MAPK pathways. These findings are remarkable when considering the use of commercially available CV by diabetic patients who also suffer from cancer or microbial infections.     

Upregulation of Glut-4 and PPAR gamma by an isoflavone from Pterocarpus marsupium on L6 myotubes: a possible mechanism of action

The purpose of the present study is to analyse the influence of Pterocarpus marsupium methanolic extract and isolated Pterocarpus marsupium isoflavone on a battery of cellular targets Glut-4, PPAR gamma and PI3 kinase. Pterocarpus marsupium is an anti-diabetic plant indigenous to South India. Sequential extraction performed with different solvents were analysed for glucose uptake activity at each step. Fraction-9 showing maximum glucose activity on glucose uptake was purified by column chromatography and the structure was elucidated as 7-O-alpha-L-rhamnopyranosyl oxy-4'-methoxy-5-hydroxy isoflavone using NMR and mass spectroscopy. The significant glucose uptake showed by Pterocarpus marsupium crude and pure was comparable with insulin and rosiglitazone. Elevation of Glut-4 and PPARgamma gene expression in parallel with glucose uptake supported the in vitro glucose uptake activity of Pterocarpus marsupium methanolic extract and Pterocarpus marsupium isoflavone. The inhibitory effect of cycloheximide on Pterocarpus marsupium methanolic extract and Pterocarpus marsupium isoflavone-mediated glucose uptake suggested that new protein synthesis is required for elevated Glut-4 protein expression. PI3 kinase plays an important role in glucose transport and activated by Pterocarpus marsupium methanolic extract but not the isolated pure isoflavone. Therefore, we postulate that the isoflavone from Pterocarpus marsupium may activate glucose transport by a PI3 kinase independent pathway, which require further analysis.     

Effect of ginsenosides Rg3 and Re on glucose transport in mature 3T3-L1 adipocytes

Ginsenosides, the active component of Panax ginseng, have been shown to evidence a variety of biological activities associated with hyperglycemia, obesity and type 2 diabetes mellitus. This study evaluated the effects of the ginsenosides, Rg3 and Re, on glucose uptake and the glucose transport system in mature 3T3‐L1 cells. The results demonstrated that the glucose uptake of ginsenosides Rg3 and Re at concentrations of 1–10 µM significantly increased by approximately ～10% and ～12%, respectively. Furthermore, the glucose transporter 4 (GLUT4) mRNA expression of ginsenosides Rg3 and Re at 10 µM was increased by approximately ～1.73 and 1.43 fold, respectively. It was further confirmed in a series of experiments that ginsenosides Rg3 and Re stimulated the mRNA expression of insulin receptor substrate (IRS‐1) and the expression of phosphatidylinositol 3‐kinase (PI3K)‐110α protein, which is involved in downstream events in the insulin signaling pathway. These findings demonstrate that ginsenosides Rg3 and Re may stimulate glucose uptake via the PI3K pathways involving IRS‐1. Further, our results suggest that both of these ginsenosides might prove useful as effective antidiabetic and antihyperglycemic agents. Copyright © 2010 John Wiley & Sons, Ltd.     

Water extracts from Momordica charantia increase glucose uptake and adiponectin secretion in 3T3-L1 adipose cells

To examine the effects of Momordica charantia on glucose uptake and adiponectin secretion in adipose cells, 3T3-L1 adipocytes were treated with three concentrations (0.2, 0.3 and 0.4mg/ml) of water and ethanol extracts of Momordica charantia fruit and seeds alone and in combination with either 0.5nM or 50nM insulin. The treatment combination of 0.2mg/ml water extract and 0.5nM insulin was associated with significant (p<0.05) increases in glucose uptake (61%) and adiponectin secretion (75%) over control levels. The ethanol extract was not associated with an increase in glucose uptake; however, a dose-dependent decrease in basal glucose uptake and insulin-mediated glucose uptake was observed with the ethanol extract in combination with 50nM insulin. In the absence of insulin, no effects on glucose uptake were observed in adipocytes exposed to the water extracts whereas the highest concentration (0.4mg/ml) of the ethanol extract was associated with a significant (p<0.05) decrease in glucose uptake relative to controls. The present results indicate that water-soluble component(s) in Momordica charantia enhance the glucose uptake at sub-optimal concentrations of insulin in 3T3-L1 adipocytes, which is accompanied by and may be a result of increased adiponectin secretion from the 3T3-L1 adipocytes.     

Antihyperglycemic effect of puerarin in streptozotocin-induced diabetic rats

The antihyperglycemic action of puerarin, purified from the roots of Pueraria lobata, was investigated in streptozotocin-induced diabetic rats (STZ-diabetic rats). Bolus intravenous injection of puerarin decreased the plasma glucose concentrations in a dose-dependent manner in STZ-diabetic rats. Similar treatment with puerarin also decreased the plasma glucose in normal rats, although the effect was not as great as that in STZ-diabetic rats. Puerarin at the effective dose (15.0 mg/kg) significantly attenuated the increase of plasma glucose induced by an intravenous glucose challenge test in normal rats. In the isolated soleus muscle of STZ-diabetic rats, puerarin enhanced the uptake of radioactive glucose in a concentration-dependent manner. Moreover, the mRNA and protein levels of the subtype 4 form of glucose transporter (GLUT4) in soleus muscle were increased after repeated intravenous administration of puerarin in STZ-diabetic rats for 3 days. These results suggest that puerarin can increase the glucose utilization to lower plasma glucose in diabetic rats lacking insulin.     

基于GLUT4转位的中药及活性成分改善胰岛素抵抗研究进展

对中药通过调控葡萄糖转运蛋白4(glucose transporter 4,GLUT4)转位而改善胰岛素抵抗(insulin resistance,IR)的研究进展进行总结.GLUT4转位研究对IR的机制研究以及药物作用靶点的选择都具有重要意义.本文主要从磷脂酰肌醇3-激酶/非典型蛋白激酶C/蛋白激酶B(PI3K/ PKC/ Akt),AMP活化蛋白激酶(AMP-activated protein kinase,AMPK)等关键途径,介绍各种中药活性成分、单方及复方通过调控GLUT4转位对IR的改善作用及机制,为防治IR中药的临床合理使用及新药开发提供参考.     

Effects of hypocrellin A on expression of vascular endothelial growth factor and endothelin-1 in human umbilical endothelial cells

Increased endothelin-1 (ET-1), vascular endothelial growth factor (VEGF) and activation of protein kinase C (PKC) are co-contributors to endothelial hyperpermeability in diabetes. Several lines of evidence have suggested a hypothesis that activation of specific PKC isoforms are the causative factor in ET-1 and VEGF mediated endothelial dysfunction. In the present study, we tested this hypothesis with hypocrellin A, a naturally occurring PKC inhibitor from a Chinese plant. Human umbilical vein endothelial cells (HUVECs) were incubated with 20 mM glucose in both the presence and absence of hypocrellin A, after which, the protein expression and release of VEGF and mRNA expression and release of ET-1 were measured. VEGF and ET-1 were released into the medium and expressions of VEGF protein and ET-1 mRNA were significantly increased in HUVECs incubated with 20 mM glucose. Hypocrellin A (150 nM) significantly decreased VEGF release (117 +/- 3 vs. 180 +/- 11 pg/mg, p < 0.05) and VEGF protein expression (from 130 +/- 14% to 88 +/- 18.5%, p < 0.05). ET-1 release was also reduced in hypocrellin A treated HUVECs (63.3 +/- 9.9 vs. 75.2 +/- 12.6 ng/mg). Hypocrellin A significantly reversed the effect of high glucose on ET-1 mRNA expression (p < 0.05). The results revealed that PKC activation plays a pivotal role in VEGF and ET-1 mediated endothelial permeability. The naturally occurring compound hypocrellin A may be a potentially novel treatment for endothelial dysfunction in diabetes.     

Ginsenoside Rg1 promotes glucose uptake through activated AMPK pathway in insulin-resistant muscle cells

Ginsenoside Rg1, a protopanaxatriols saponin, is one of the major active constituents from Panax ginseng and possesses various biological activities. A recent study reported that insulin resistance in skeletal muscle is a major contributor to the development of type 2 diabetes mellitus (T2DM). We examined the effects of ginsenoside Rg1 on glucose uptake and the associated molecular mechanisms of the glucose transport system in insulin-resistant muscle cells. The insulin resistance of the muscle cell was induced by treatment of differentiated C2C12 cells with chronic insulin. The results showed that chronic treatment of insulin resulted in reduced glucose uptake in the muscle cells. The treatment of ginsenoside Rg1 significantly enhanced glucose uptake in the differentiated muscle cells and the relative abundance of GLUT4 through the adenosine-monophosphate-activated protein kinase pathway. These results suggest that ginsenoside Rg1 improved the insulin resistance in C2C12 muscle cells, which might be useful for prevention of T2DM and metabolic syndromes.     

Mulberry leaf extract stimulates glucose uptake and GLUT4 translocation in rat adipocytes

Mulberry (Morus alba L.) leaf tea is promoted for its health benefits and the control of diabetes in Asian nations. The blood glucose lowering activity of mulberry leaf extract (MA) has been proven; however, the molecular basis underlying this effect remains unclear. The aim of the present work is to elucidate its mechanism of the antihyperglycemic action, by examining the effect of MA on glucose uptake and the translocation of glucose transporter 4 protein (GLUT4) to the plasma membrane of adipocytes isolated from diabetic rats. The incubation of adipocytes with 5-45 μg/ml MA resulted in 31-54% increase of glucose uptake in a dose-dependent manner. This glucose uptake enhancing effect was inhibited by the phosphoinositol 3-kinase (PI3-K) inhibitor, wortmannin (100 nM). The GLUT4 protein on the plasma membrane fraction of adipocytes was markedly increased after treatment with 15 μg/ml MA extract. Interestingly, gallic acid, one of the phenolic compounds found in MA extract, increased glucose uptake and enhanced the translocation of GLUT4 at concentrations comparable to the amount of gallic acid in the effective concentration ranges of MA. Thus, it is likely that gallic acid contributes, at least in part, to its antihyperglycemic activity. The present results suggest that the antihyperglycemic action of MA is mediated by increasing glucose uptake via the activation of PI3-K signaling pathway and translocation of GLUT4 to the plasma membrane. These findings are the first molecular evidence supporting the mulberry tea as herbal medicine for diabetic patients.     

Scoparia dulcis (SDF7) endowed with glucose uptake properties on L6 myotubes compared insulin

Aim of the study

Insulin stimulates glucose uptake and promotes the translocation of glucose transporter 4 (Glut 4) to the plasma membrane on L6 myotubes. The aim of this study is to investigate affect of Scoparia dulcis Linn water extracts on glucose uptake activity and the Glut 4 translocation components (i.e., IRS-1, PI 3-kinase, PKB/Akt2, PKC and TC 10) in L6 myotubes compared to insulin.

Materials and methods

Extract from TLC fraction-7 (SDF7) was used in this study. The L6 myotubes were treated by various concentrations of SDF7 (1 to 50 μg/ml) and insulin (1 to 100 nM). The glucose uptake activities of L6 myotubes were evaluated using 2-Deoxy-D-glucose uptake assay in with or without fatty acid-induced medium. The Glut 4 translocation components in SDF7-treated L6 myotubes were detected using immunoblotting and quantified by densitometry compared to insulin. Plasma membrane lawn assay and glycogen colorimetry assay were carried out in SDF7- and insulin-treated L6 myotubes in this study.

Results

Here, our data clearly shows that SDF7 possesses glucose uptake properties on L6 myotubes that are dose-dependent, time-dependent and plasma membrane Glut 4 expression-dependent. SDF7 successfully stimulates glucose uptake activity as potent as insulin at a maximum concentration of 50 μg/ml at 480 min on L6 myotubes. Furthermore, SDF7 stimulates increased Glut 4 expression and translocation to plasma membranes at equivalent times. Even in the insulin resistance stage (free fatty acids-induced), SDF7-treated L6 myotubes were found to be more capable at glucose transport than insulin treatment.

Conclusions

Thus, we suggested that Scoparia dulcis has the potential to be categorized as a hypoglycemic medicinal plant based on its good glucose transport properties.     

Mechanisms of Panax ginseng in preventing rat pheochromocytoma cells from apoptosis

Aims of the study

Although ginseng root possesses dominant central therapeutic effects and has recently undergone investigations for treating different neuronal diseases, most of its mechanisms are still unknown. Therefore, the neuroprotective mechanisms of ginseng were studied.

Materials and methods

The protection afforded by different methanol extracts of Panax ginseng (PG) was tested in a serum deprivation-induced apoptotic model using neuronal-like pheochromocytoma (PC12) cells. An MTT assay, annexin V-FITC staining, and Western blots were, respectively, applied to identify the viability of cells, the apoptotic form of cell death, and the activity of antiapoptotic signaling.

Results

The known antiapoptotic PI3-K/Akt and MEK/ERK pathways in this system were ruled out due to failure of LY 294002 and PD 98059 to block the protection by PG. A protein kinase A (PKA) inhibitor was found to block the protection by PG and PG-induced CREB phosphorylation, suggesting that the PKA/CREB pathway mediates the protective effect of PG. Downregulation of classical and novel PKCs failed to block the protection by PG, while an atypical PKC inhibitor blocked protection by PG.

Conclusions

PKA and atypical PKC are important for the protection afforded by PG in preventing serum deprivation-induced PC12 cell apoptosis.     

Cardamonin stimulates glucose uptake through translocation of glucose transporter-4 in L6 myotubes

Glucose transporter-4 (GLUT4) is a transmembrane protein that plays a major role in insulin-mediated glucose transport in muscle and adipocytes. For glucose transport to occur, the GLUT4 protein needs to be translocated from the intracellular pool to the plasma membrane, and certain compounds may enhance this process. The present study investigated the promotion of glucose uptake in differentiated L6 myotubes by cardamonin, isolated from Alpinia katsumadai. Cardamonin increased translocation of GLUT4 to the plasma membrane in L6 cells, but did not activate protein kinase C ζ/λ, Akt, or AMP-activated protein-kinase, all of which are known to regulate GLUT4 translocation. The glucose-uptake-promoting activity of cardamonin was not lowered by treatment with a phosphatidylinositol 3'-kinase inhibitor. These results suggest that cardamonin is a promising active compound for maintaining glucose homeostasis, and that it acts via an unknown mechanism that does not involve activation of the downstream insulin signal and AMP-activated protein kinase.     

Polymethoxyflavonoids Tangeretin and Nobiletin Increase Glucose Uptake in Murine Adipocytes

Tangeretin and nobiletin are polymethoxyflavonoids that are contained in citrus fruits. Polymethoxyflavonoids are reported to have several biological functions including anti‐inflammatory, anti‐atherogenic, or anti‐diabetic effects. However, whether polymethoxyflavonoids directly affect glucose uptake in tissues is not well understood. In the current study, we investigated whether tangeretin and nobiletin affect glucose uptake in insulin target cells such as adipocytes. We observed that treatment with tangeretin or nobiletin significantly increased the uptake of [³H]‐deoxyglucose in differentiated 3T3‐F442A adipocytes in a concentration‐dependent manner. Data showed that phosphatidyl inositol 3 kinase, Akt1/2, and the protein kinase A pathways were involved in the increase in glucose uptake induced by polymethoxyflavonoids. These data suggest that the anti‐diabetic action of polymethoxyflavonoids is partly exerted via these signaling pathways in insulin target tissues. Copyright © 2012 John Wiley & Sons, Ltd.