Momordica charantia fruit juice stimulates glucose and amino acid uptakes in L6 myotubes

The fruit of Momordica charantia (family: Cucurbitacea) is used widely as a hypoglycaemic agent to treat diabetes mellitus (DM). The mechanism of the hypoglycaemic action of M. charantia in vitro is not fully understood. This study investigated the effect of M. charantia juice on either ³H-2-deoxyglucose or N-methyl-amino-a-isobutyric acid (¹⁴C-Me-AIB) uptake in L6 rat muscle cells cultured to the myotube stage. The fresh juice was centrifuged at 5000 rpm and the supernatant lyophilised. L6 myotubes were incubated with either insulin (100 nM), different concentrations (1–10 g ml^–1) of the juice or its chloroform extract or wortmannin (100 nM) over a period of 1–6 h. The results were expressed as pmol min^–1 (mg cell protein)^–1, n= 6–8 for each value. Basal ³H-deoxyglucose and ¹⁴C-Me-AIB uptakes by L6 myotubes after 1 h of incubation were (means ± S.E.M.) 32.14 ± 1.34 and 13.48 ± 1.86 pmol min^–1 (mg cell protein)^–1, respectively. Incubation of L6 myotubes with 100 nM insulin for 1 h resulted in significant (ANOVA, p < 0.05) increases in ³H-deoxyglucose and ¹⁴C-Me-AIB uptakes. Typically, ³H-deoxyglucose and ¹⁴C-Me-AIB uptakes in the presence of insulin were 58.57 ± 4.49 and 29.52 ± 3.41 pmol min^–1 (mg cell protein^–1), respectively. Incubation of L6 myotubes with three different concentrations (1, 5 and 10 g ml^–1) of either the lyophilised juice or its chloroform extract resulted in time-dependent increases in ³H-deoxy-D-glucose and ¹⁴C-Me-AIB uptakes, with maximal uptakes occurring at a concentration of 5 g ml^–1. Incubation of either insulin or the juice in the presence of wortmannin (a phosphatidylinositol 3-kinase inhibitor) resulted in a marked inhibition of ³H-deoxyglucose by L6 myotubes compared to the uptake obtained with either insulin or the juice alone. The results indicate that M. charantia fruit juice acts like insulin to exert its hypoglycaemic effect and moreover, it can stimulate amino acid uptake into skeletal muscle cells just like insulin. (Mol Cell Biochem 261: 99–104, 2004)     

Isoleucine, a potent plasma glucose-lowering amino acid, stimulates glucose uptake in C2C12 myotubes

To examine which branched-chain amino acids affect the plasma glucose levels, we investigated the effects of leucine, isoleucine, and valine (0.3 g/kg body weight p.o.) in normal rats using the oral glucose tolerance test (OGTT, 2 g/kg). A single oral administration of isoleucine significantly reduced plasma glucose levels 30 and 60 min after the glucose bolus, whereas administration of leucine and valine did not produce a significant decrease. Oral administration of valine significantly enhanced the plasma glucose level at 30 min after the glucose administration and leucine had a similar effect at 120 min. At each measurement timepoint, the insulin levels of the treated groups were lower than that of the control group. We then investigated the effects of leucine, isoleucine or valine at the same concentration (1 mM) on glucose metabolism in C(2)C(12) myotubes in the absence of insulin. Glucose consumption was elevated by 16.8% in the presence of 1 mM isoleucine compared with the control. Conversely, 1 mM leucine or valine caused no significant changes in glucose consumption in the C(2)C(12) myotubes. The 2-deoxyglucose uptake of C(2)C(12) myotubes significantly increased upon exposure to 1-10 mM isoleucine and 5-10 mM leucine. However, isoleucine caused no significant difference in glycogen synthesis in C(2)C(12) myotubes, although leucine and valine caused a significant increase in intracellular glycogen compared with the control. The isoleucine effect on glucose uptake was mediated by phosphatidylinositol 3-kinase (PI3K), but was independent of mammalian target of rapamycin (mTOR). These results suggest that isoleucine stimulates the insulin-independent glucose uptake in skeletal muscle cells, which may contribute to the plasma glucose-lowering effect of isoleucine in normal rats.     

Identification of an amino acid determinant of pH regiospecificity in a seed lipoxygenase from Momordica charantia

Lipoxygenases (LOX) form a heterogeneous family of lipid peroxidizing enzymes, which catalyze specific dioxygenation of polyunsaturated fatty acids. According to their positional specificity of linoleic acid oxygenation plant LOX have been classified into linoleate 9- and linoleate 13-LOX and recent reports identified a critical valine at the active site of 9-LOX. In contrast, more bulky phenylalanine or histidine residues were found at this position in 13-LOX. We have recently cloned a LOX-isoform from Momordica charantia and multiple amino acid alignments indicated the existence of a glutamine (Gln599) at the position were 13-LOX usually carry histidine or phenylalanine residues. Analyzing the pH-dependence of the positional specificity of linoleic acid oxygenation we observed that at pH-values higher than 7.5 this enzyme constitutes a linoleate 13-LOX whereas at lower pH, 9-H(P)ODE was the major reaction product. Site-directed mutagenesis of glutamine 599 to histidine (Gln599His) converted the enzyme to a pure 13-LOX. These data confirm previous observation suggesting that reaction specificity of certain LOX-isoforms is not an absolute enzyme property but may be impacted by reaction conditions such as pH of the reaction mixture. We extended this concept by identifying glutamine 599 as sequence determinant for such pH-dependence of the reaction specificity. Although the biological relevance for this alteration switch remains to be investigated it is of particular interest that it occurs at near physiological conditions in the pH-range between 7 and 8.     

HPLC-ELSD测定苦瓜皂甙的含量

本文用HPLCELSD法测定苦瓜中皂甙B的含量。色谱条件:色谱柱ZorbaxSBC18,4.6×150mm;检测器ELSD(EvaporativeLightScatteringDetector);柱温30℃;流动相:甲醇水(70:30);流速1.0mL/min,N2压力:2.0×105Pa。皂甙B的保留时间在为7.295min,在浓度为0.1058～5.290μg/μL之间有很好的线性关系(R2=0.9918)。平均加标回收率为100.27%(n=3),RSD=2.83%。实验结果表明此方法可以作为苦瓜皂甙的定量测定方法     

Regulation of uterine and umbilical amino acid uptakes by maternal amino acid concentrations

We tested the hypothesis that decreased fetal amino acid (AA) supply, produced by maternal hypoaminoacidemia (low AA) during hyperglycemia (HG), is reversible with maternal AA infusion and regulates fetal insulin concentration ([I]). We measured net uterine and umbilical AA uptakes during maternal HG/low AA concentration ([AA]) and after maternal intravenous infusion of a mixed AA solution. After 5 days HG, all maternal [AA] except glycine were decreased >50%, particularly essential [AA] (P < 0.00005). Most fetal [AA] also were decreased, especially branched-chain AA (P < 0.001). Maternal AA infusion increased net uterine uptakes of Val, Leu, Ile, Met, and Ser and net umbilical uptakes of Val, Leu, Ile, Met, Phe, and Arg but did not change net uteroplacental uptake of any AA. Fetal [I] increased 55 +/- 14%, P < 0.001, with correction of fetal [AA], despite the lack of change in fetal glucose concentration. Thus generalized maternal hypoaminoacidemia decreases uterine and umbilical uptakes of primarily the essential AA and decreases fetal branched-chain [AA]. These changes are reversed with correction of maternal [AA], which also increases fetal [I].     

Inheritance of gynoecism in bitter gourd (Momordica charantia L.)

The inheritance of sex expression in cucumber (Cucumis sativus) and other cucurbits is well documented; however, the genetics of female sex (gynoecism) expression in bitter gourd (Momordica charantia) has not been described. Inheritance of gynoecism in bitter gourd was studied in a 100% gynoecious line (Gy263B). The F(2) and testcross segregation data revealed that gynoecism in Gy263B is under the control of a single, recessive gene. Following the gene nomenclature of cucurbits, it is proposed that the gene symbol, gy-1, be assigned for the expression of gynoecism in bitter gourd.     

Berberine-stimulated glucose uptake in L6 myotubes involves both AMPK and p38 MAPK

Berberine is a plant alkaloid used in traditional Chinese medicine and has been reported to have antihyperglycemic activity in NIDDM patients. However, the molecular basis for this action is yet to be elucidated. Here we investigate the effects and signaling pathways of berberine on L6 rat skeletal muscles. Our study demonstrates that berberine stimulates glucose uptake in a time- and dose-dependent manner. Intriguingly, berberine-stimulated glucose uptake does not vary as insulin concentration increases, and could not be blocked by the PI 3-kinase inhibitor wortmannin. Berberine only weakly stimulates the phosphorylation of Akt/PKB, a key molecule in the insulin signaling pathway, but strongly promotes the phosphorylation of AMPK and p38 MAPK. The effects of berberine are not a result of pro-oxidant action, but a consequence of an increased cellular AMP:ATP ratio. Moreover, berberine-stimulated glucose uptake is inhibited by the AMPK inhibitor Compound C and the p38 MAPK inhibitor SB202190. Inhibition of AMPK reduces p38 MAPK phosphorylation, suggesting that AMPK lies upstream of p38 MAPK. These results suggest that berberine circumvents insulin signaling pathways and stimulates glucose uptake through the AMP-AMPK-p38 MAPK pathway, which may account for the antihyperglycemic effects of this drug.     

The complete amino acid sequence of ribonuclease from the seeds of bitter gourd (Momordica charantia)

The complete amino acid sequence of ribonuclease (RNase MC) from the seeds of bitter gourd (Momordica charantia) has been determined. This has been achieved by the sequence analysis of peptides derived by enzymatic digestion with trypsin, lysylendopeptidase, and chymotrypsin, as well as by chemical cleavage with cyanogen bromide. The protein contains 191 amino acid residues and has a calculated molecular mass of 21,259 Da. Comparison of this sequence with sequences of the fungal RNases, RNase T2, and RNase Rh, revealed that there are highly conserved residues at positions 32-38 (TXHGLWP) and 81-92 (FWXHEWXKHGTC). Furthermore, the sequence of RNase MC was found to be homologous to those of Nicotiana alata S-glycoproteins involved in self-incompatibility sharing 41% identical residues.     

Interaction of phytochemicals with hypoglycemic drugs on glucose uptake in L6 myotubes

The present study analyses the effect of eugenol, arecoline and vanillic acid alone and in combination with two oral hypoglycemic drugs (OHD), namely, metformin and 2,4-thiazolodinedione (THZ), on 2-deoxyglucose (2DG) uptake in L6 myotubes. 2DG uptake in L6 myotubes was determined using an enzymatic assay developed by Yamamoto et al. (2006). Lipid content inside the cells has been estimated with oil red O assay. The absorption, distribution, metabolism, and excretion (ADME) and drug likeness properties of these phytochemicals are estimated using software QikProp^®. All the three phytochemicals enhance 2DG uptake both in time- and dose-dependent manner. Eugenol and arecoline enhances 2DG uptake synergistically with both the OHD; whereas vanillic acid showing partly synergy with THZ and antagonistic activity with metformin on 2DG uptake. Eugenol and arecoline significantly increase the expressions of the glucose transporter type 4 (GLUT4) and phosphoinositide 3-kinase (PI3K) genes, but not the peroxisome proliferator-activated receptor (PPAR) gamma. Whereas vanillic acid does not has any significant effect on the expressions of these genes, the ADME results indicate that these phytochemicals are satisfying all the conditions to have a good oral bioavailability. These findings suggest that these phytochemicals can replace the commercial drugs in part, which could lead to a reduction in toxicity and side effects caused by the later as well as reduce the secondary complications.     

Acacetin enhances glucose uptake through insulin-independent GLUT4 translocation in L6 myotubes

BackgroundLowering blood glucose levels by increasing glucose uptake in insulin target tissues, such as skeletal muscle and adipose tissue, is one strategy to discover and develop antidiabetic drugs from natural products used as traditional medicines.PurposeOur goal was to reveal the mechanism and activity of acacetin (5,7-dihydroxy-4′-methoxyflavone), one of the major compounds in Agastache rugose, in stimulating glucose uptake in muscle cells.MethodsTo determine whether acacetin promotes GLUT4-dependent glucose uptake in cultured L6 skeletal muscle cells, we performed a [¹⁴C] 2-deoxy-_D-glucose (2-DG) uptake assay after treating differentiated L6-GLUT4myc cells with acacetin.ResultsAcacetin dose-dependently increased 2-DG uptake by enhancing GLUT4 translocation to the plasma membrane. Our results revealed that acacetin activated the CaMKII-AMPK pathway by increasing intracellular calcium concentrations. We also found that aPKCλ/ζ phosphorylation and intracellular reactive oxygen species (ROS) production were involved in acacetin-induced GLUT4 translocation. Moreover, acacetin-activated AMPK inhibited intracellular lipid accumulation and increased 2-DG uptake in HepG2 cells.ConclusionTaken together, these results suggest that acacetin might be useful as an antidiabetic functional ingredient. Subsequent experiments using disease model animals are needed to verify our results.     

苦瓜化学成分和生物活性的研究进展

本文就近年来国内外对苦瓜的研究情况进行综述,主要概述了其化学成分、生物活性(药理活性和抗虫作用)的进展情况。     

The hexosamine biosynthetic pathway and glucose-induced down regulation of glucose transport in L6 myotubes

Based on experiments in cultured adipocytes, it has been proposed that glucose-induced down regulation of glucose transport is mediated by the conversion of fructose-6-phosphate to glucosamine-6-phosphate via the first and rate-determining enzyme of the hexasamine biosynthetic pathway, glutamine: fructose-6-phosphate amidotransferase (glutamine hexosephosphate aminotransferase). Evidence for this assertion was: (a) l-glutamine, the provider group for the aminotransferase was essential; (b) two inhibitors of glutamine hexosephosphate aminotransferase, 6-diazo-5-oxonorleucine (l form) and azaserine, blocked glucose-induced down regulation of glucose transport; (c) azaserine inhibited the activity of the aminotransferase, (d) glucosamine, which enters the hexosamine pathway distal to this enzyme was 40-times more potent than glucose; and (e) azaserine was unable to block the effect of glucosmaine. Since muscle is quantitatively much more important than adipose tissue for whole body glucose utilization, we sought to determine if the hexosamine pathway was involved in glucose-induced down regulation of glucose transport in L₆ myotubes. Glucose was effective, both in the presence and absence of glutamine in the incubation media. Glucosamine was also effective but was as equipotent as glucose. Small amounts of glutamine hexosephosphate aminotransferase were present in the L₆ myotubes and although the leucine derivative (20 μM)_ inhibited the enzyme, it did not impair glucose-induced down regulation of glucose transport. Total GLUT-1 levels were similar when the cells were incubated in the absence or presence of 5 mM glucose or glucosamine although glucosamine was associated with a marked increase in a lower molecular weight band. These results do not suggest that the hexosamine biosynthetic pathway is involved in glucose-induced down regulation of glucose transport in L₆ myotubes. Thus, this phenomenon is regulated differently in muscle and fat.     

Inhibition of increases in blood glucose and serum neutral fat by Momordica charantia saponin fraction

Focusing on a functional component of Momordica charantia, saponin, we investigated its effects on serum glucose and neutral fat levels. Saponin was extracted as a butanol-soluble fraction (saponin fraction) from hot blast-dried Momordica charantia powder. The disaccharidase-inhibitory activity and the pancreatic lipase-inhibitory activity of the saponin fraction were measured, and in vivo sugar- and lipid-loading tests were performed. The saponin fraction inhibited disaccharidase activity and elevation of the blood glucose level after sucrose loading. The fraction also markedly inhibited pancreatic lipase activity and elevation of the serum neutral fat level after corn oil loading. Based on these findings, the main active component related to the anti-diabetic effect of Momordica charantia is present in the butanol fraction, and it may be saponin. The blood glucose and serum neutral fat-lowering effects of Momordica charantia were closely associated with its inhibitory activity against disaccharidase and pancreatic lipase.     

Genome-wide mining of potentially-hypervariable microsatellites and validation of markers in Momordica charantia L.

Genetica - Relatively large number of bitter melon microsatellite markers have been reported; however, only few resulted in successful PCR amplification and a small fraction shown polymorphisms....     

Catalytic synthesis and antioxidant activity of sulfated polysaccharide from Momordica charantia L.

Sulfated derivatives of polysaccharide from Momordica charantia L. (MCPS) with different degree of sulfation (DS) were synthesized by chlorosulfonic acid method with ionic liquids as solvent. Fourier transform infrared spectra and ¹³C nuclear magnetic resonance spectra indicated that C‐6 substitution was predominant in MCPS compared with the C‐2 position. Compared with the native polysaccharide from Momordica charantia L. (MCP), MCPS exhibited more excellent antioxidant activities in vitro, which indicated that sulfated modification could enhance antioxidant activities of MCP. Furthermore, high DS and moderate molecular weight could improve the antioxidant activities of polysaccharide. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 210–215, 2014.