Aqueous bark extracts of terminalia arjuna stimulates insulin release, enhances insulin action and inhibits starch digestion and protein glycation in vitro

Scientific evidence for the effects of Terminalia arjuna on diabetes mellitus is lacking. This study investigated the anti-diabetic efficacy and mode of action of the bark extract of Terminalia arjuna. Insulin-release from BRIN-BD11 cells was assessed in the absence or presence of T. arjuna extracts and modulators of insulin-secretion. Insulin concentration and intracellular calcium ([Ca2+]i) were measured by radioimmunoassay and FLEXstationTM respectively. Adipocyte glucose-uptake with 3T3-L1 fibroblasts, starch digestion with α–amylase and protein glycation were assessed in vitro. Terminalia arjuna extract stimulated insulin-release alone (p<0.001), in combination with known modulators but not without extracellular Ca2+. It increased intracellular calcium but had no effects on depolarised cells. Glucose-uptake was enhanced in the presence of T. arjuna (P< 0.001). At higher concentrations, the extract decreased starch digestion and inhibited protein glycation (p<0.001). Terminalia arjuna extract possesses antidiabetic potential and may provide new opportunities for the treatment of diabetes

In vitro and in vivo insulinotropic properties of the multifunctional frog skin peptide hymenochirin-1B: a structure–activity study

Hymenochirin-1b (Hym-1B; IKLSPETKDNLKKVLKGAIKGAIAVAKMV.NH2) is a cationic, α-helical amphibian host-defense peptide with antimicrobial, anticancer, and immunomodulatory properties. This study investigates the abilities of the peptide and nine analogues containing substitutions of Pro5, Glu6, and Asp9 by either l-lysine or d-lysine to stimulate insulin release in vitro using BRIN-BD11 clonal β cells or isolated mouse islets and in vivo using mice fed a high-fat diet to produce obesity and insulin resistance. Hym-1B produced a significant and concentration-dependent increase in the rate of insulin release from BRIN-BD11 cells without cytotoxicity at concentrations up to 1 µM with a threshold concentration of 1 nM. The threshold concentrations for the analogues were: [P5K], [E6K], [D9K], [P5K, E6K] and [E6K, D9k] 0.003 nM, [E6K, D9K] and [D9k] 0.01 nM, [P5K, D9K] 0.1 nM and [E6k] 0.3 nM. All peptides displayed cytotoxicity at concentrations ≥1 µM except the [P5K] and [D9k] analogues which were non-toxic at 3 µM. The potency and maximum rate of insulin release from mouse islets produced by the [P5K] peptide were significantly greater than produced by Hym-1B. Neither Hym-1B nor the [P5K] analogue at 1 µM concentration had an effect on membrane depolarization or intracellular Ca2+. The [P5K] analogue (1 µM) produced a significant increase in cAMP concentration in BRIN-BD11 cells and stimulated GLP-1 secretion from GLUTag cells. Down-regulation of the protein kinase A pathway by overnight incubation with forskolin completely abolished the insulin-releasing effects of [P5K]hym-1B. Intraperitoneal administration of the [P5K] and [D9k] analogues (75 nmol/kg body weight) to high-fat-fed mice with insulin resistance significantly enhanced glucose tolerance with a concomitant increase in insulin secretion. We conclude that [P5K]hym-1B and [D9k]hym-1B show potential for development into anti-diabetic agents

N-terminal glycation of cholecystokinin-8 abolishes its insulinotropic action on clonal pancreatic B-cells

AbstractMonoglycated cholecystokinin octapeptide (Asp1-glucitol CCK-8) was prepared under hyperglycaemic reducing conditions and purified by reverse phase-high performance liquid chromatography. Electrospray ionisation mass spectrometry and automated Edman degradation demonstrated that CCK-8 was glycated specifically at the amino-terminal Asp1 residue. Effects of Asp1-glucitol CCK-8 and CCK-8 on insulin secretion were examined using glucose-responsive clonal BRIN-BD11 cells. In acute (20 min) incubations, 10−10 mol/l CCK-8 enhanced insulin release by 1.2–1.5-fold at 5.6–11.1 mmol/l glucose. The stimulatory effect induced by 10−10 mol/l CCK-8 was abolished following glycation. At 5.6 mmol/l glucose, CCK-8 at concentrations ranging from 10−11 to 10−7 mol/l induced a significant 1.6–1.9-fold increase in insulin secretion. Insulin output in the presence of Asp1-glucitol CCK-8 over the concentration range 10−11–10−7 mol/l was decreased by 21–35% compared with CCK-8, and its insulinotropic action was effectively abolished. Asp1-glucitol CCK-8 at 10−8 mol/l also completely blocked the stimulatory effects of 10−11–10−8 mol/l CCK-8. These data indicate that structural modification by glycation at the amino-terminal Asp1 residue effectively abolishes and/or antagonises the insulinotropic activity of CCK-8

Esculentin-2CHa(1–30) and its analogues: stability and mechanisms of insulinotropic action

The insulin-releasing effects, cellular mechanisms of action and anti-hyperglycaemic activity of 10 analogues of esculentin-2CHa lacking the cyclic C-terminal domain (CKISKQC) were evaluated. Analogues of the truncated peptide, esculentin-2CHa(1–30), were designed for plasma enzyme resistance and increased biological activity. Effects of those analogues on insulin release, cell membrane integrity, membrane potential, intracellular Ca2+ and cAMP levels were determined using clonal BRIN-BD11 cells. Their acute effects on glucose tolerance were investigated using NIH Swiss mice. d-Amino acid substitutions at positions 7(Arg), 15(Lys) and 23(Lys) and fatty acid (l-octanoate) attachment to Lys at position 15 of esculentin-2CHa(1–30) conveyed resistance to plasma enzyme degradation whilst preserving insulin-releasing activity. Analogues, [d-Arg7,d-Lys15,d-Lys23]-esculentin-2CHa(1–30) and Lys15-octanoate-esculentin-2CHa(1–30), exhibiting most promising profiles and with confirmed effects on both human insulin-secreting cells and primary mouse islets were selected for further analysis. Using chemical inhibition of adenylate cyclase, protein kinase C or phospholipase C pathways, involvement of PLC/PKC-mediated insulin secretion was confirmed similar to that of CCK-8. Diazoxide, verapamil and Ca2+ omission inhibited insulin secretion induced by the esculentin-2CHa(1–30) analogues suggesting an action on KATP and Ca2+ channels also. Consistent with this, the analogues depolarised the plasma membrane and increased intracellular Ca2+. Evaluation with fluorescent-labelled esculentin-2CHa(1–30) indicated membrane action, with internalisation; however, patch-clamp experiments suggested that depolarisation was not due to the direct inhibition of KATP channels. Acute administration of either analogue to NIH Swiss mice improved glucose tolerance and enhanced insulin release similar to that observed with GLP-1. These data suggest that multi-acting analogues of esculentin-2CHa(1–30) may prove useful for glycaemic control in obesity-diabetes.</jats:p