Marie-Madeleine Loubatières-Mariani

Implication of steroid saponins and sapogenins in the hypocholesterolemic effect of fenugreek

The transformation of fenugreek subfractions, rich in steroid saponins, was studied upon their passage through the digestive tract to determine the contribution of saponins and/or diosgenin and other steroid sapogenins to the hypocholesterolemic effect of fenugreek seeds. Feces of alloxan diabetic dogs fed fenugreek subfractions were analyzed, and diosgenin, smilagenin and gitogenin were identified and measured using capillary gas chromatography/mass spectrometry. Our results show that saponins are, in part (about 57%), hydrolyzed into sapogenins in the digestive tract. It appears that saponins may be implicated, alone or together with diosgenin, in the observed hypocholesterolemic effect of fenugreek seeds in diabetic dogs.

Evidence for a glutamate receptor of the AMPA subtype which mediates insulin release from rat perfused pancreas

1 The effect of l‐glutamate has been studied on insulin secretion by the isolated perfused pancreas of the rat. The glutamate receptor subtype involved has been characterized. 2 In the presence of a slightly stimulating glucose concentration (8.3 mm), l‐glutamate (5 × 10−5−4 × 10−3 m) induced an immediate, transient and concentration‐dependent insulin response. On the other hand, in the presence of a non stimulating glucose concentration (2.8 mm), l‐glutamate (10−3 m) did not modify the basal insulin secretion. 3 The three non‐NMDA receptor agonists, kainate (10−4−10−3 m), α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA, 5 × 10−5−10−4 m) and quisqualate (5 × 10−6−5 × 10−5 m) all provoked a transient and concentration‐dependent insulin response from pancreas perfused with 8.3 mm glucose. Compared with glutamate, kainate exhibited a similar efficacy, whereas AMPA and quisqualate elicited only a 3 fold lower maximal insulin response. In contrast, NMDA (10−4−10−3 m) was ineffective. 4 An antagonist of non‐NMDA receptors, 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX; 5 × 10−5 m) totally prevented the stimulatory effect of l‐glutamate (4 × 10−4 m) and kainate (2 × 10−4 m). In contrast, the NMDA receptor antagonist, (+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5,10‐imine ((+) MK801) was without effect. 5 The insulin secretory effect of glutamate (4 × 10−4 m) was not affected by atropine (3 × 10−7 m) or tetrodotoxin (3 × 10−6 m). 6 Quisqualate at a high maximally effective concentration (4 × 10−4 m) inhibited glutamate (10−3 m) or kainate (4 × 10−4 m)‐induced insulin release. 7 This study shows that l‐glutamate stimulates insulin secretion in rat pancreas, by acting on an excitatory amino acid receptor of the AMPA subtype.

Effects of Fenugreek Seeds on Endocrine Pancreatic Secretions in Dogs

The components of fenugreek seeds were separated and analyzed to determine which fraction of the seed had hypoglycemic activity. These fractions were administered orally to normal or diabetic dogs for 8 days. The effect on blood glucose and pancreatic hormones was studied in normal dogs. The lipid extract had no effect; the defatted fraction (50.2% fibers: gum 17.7%, hemicellulose 22%, cellulose 8.3%, lignin 2.2%) lowered basal blood glucose level, plasma glucagon and somatostatin levels and reduced the orally induced hyperglycemia. The addition of this fraction to the insulin treatment resulted in a decrease of hyperglycemia and glycosuria in diabetic dogs. The results indicate that the defatted part is responsible for the antidiabetic action. However, the present study does not permit one to know whether the effects are caused by an unknown pharmacological compound or by the gastrointestinal action of fibers.

Purinergic receptors on insulin-secreting cells.

Summary— The insulin secreting B cell is fitted with the two types of purinergic receptors: P2 (for ATP and/or ADP) and P1 (for adenosine). The activation of P2 purinoceptors by ATP or ADP evokes a biphasic stimulation of insulin secretion from isolated perfused rat pancreas; this stimulation is dose‐dependent between 10−6 and 10−4 M. Non hydrolysable structural analogues are also effective, and the relative potency of various agonists (2‐methylthio ATP ≫ ATP = ADP = α, β‐methylene ATP ≫ AMP) gave evidence for a P2y purinoceptor subtype. Proposed mechanisms include both an increased Ca2+ uptake and an increased intracellular Ca2+ mobilization via the hydrolysis of polyphosphoinositides. ATP (or ADP) potentiates physiological insulin‐secreting agents (glucose and acetylcholine) and P2 purinoceptors could play a physiological role in the stimulation of insulin secretion. The activation of P1 purinoceptors (adenosine receptors) decreases insulin secretion. Using structural analogues of adenosine, the receptor was characterized as an A1 subtype; it is coupled to a pertussis toxin sensitive G protein and it inhibits adenylate cyclase. It is of physiological relevance that the B cell has the two types of purinoceptors with opposite effects. Recently, a metabolically stable structural analogue of ADP, adenosine‐5′‐0‐(2‐thiodiphosphate) or ADPßS, has been described as a potent secretory agent, effective at nanomolar concentrations on isolated perfused rat pancreas. In vivo, this substance is able to increase insulin secretion and to improve glucose tolerance after IV administration in rats and oral administration in dogs. Furthermore in streptozotocin‐induced diabetes, ADPßS retains its insulin secreting effects. These results suggest that P2y purinoceptors could be a new target for antidiabetic drugs.

Evidence for two different P2‐purinoceptors on β cell and pancreatic vascular bed

1 The effects of a 2‐substituted analogue of adenosine 5′‐triphosphate (ATP), 2‐methylthioadenosine triphosphate (2‐methylthio ATP) have been studied on insulin secretion and flow rate of the isolated pancreas of the rat, perfused in the presence of glucose (8.3 mm). 2 2‐Methylthio ATP (16.5–1650 nm) increased insulin secretion in a biphasic and concentration‐dependent manner; the kinetics were comparable to those previously obtained with ATP. A comparison of relative potency between ATP and 2‐methylthio ATP showed that 2‐methylthio ATP was 45 times more potent that ATP. 3 2‐Methylthio ATP also provoked a transient decrease of the flow rate in a concentration‐dependent manner but at concentrations (165–825 μm) about 1000 fold higher than those needed to increase insulin secretion. A comparison of relative potency between the natural derivative and 2‐methylthio ATP showed that 2‐methylthio ATP was only twice as potent as ATP. 4 These and other previous results (with phosphate‐modified analogues of ATP) provide evidence for two different types of P2‐purinoceptors on endocrine cell and vessel cells of the pancreas. A P2Y subtype, mediating an increase of insulin secretion, is present on the β cell of the pancreas. A P2X subtype, mediating vasoconstriction, is present on the vascular bed of the rat pancreas.

Comparative effects of PACAP and VIP on pancreatic endocrine secretions and vascular resistance in rat.

1 The effects of pituitary adenylate cyclase‐activating polypeptide (PACAP), vasoactive intestinal peptide (VIP) and secretin on pancreatic endocrine secretions and vascular resistance were investigated and compared in the isolated perfused pancreas of the rat. The PACAP/VIP receptor types involved have been characterized. 2 On insulin secretion, in the range 10−11 to 10−8 m, PACAP and VIP elicited a concentration‐dependent biphasic response from pancreas perfused with 8.3 mM glucose; the peptides were equipotent. In contrast, secretin was ineffective in the range 10−11 to 10−9 m; at 10−8 and 10−7 m, it induced only low and transient insulin responses. On the other hand, the peptides did not modify the basal insulin release in the presence of a non stimulating glucose concentration (2.8 mM). 3 On glucagon secretion, PACAP and VIP (10−11 to 10−8 m) but also secretin (10−9 to 10−7 m) caused a concentration‐dependent peak shaped response from pancreas perfused with 2.8 mM glucose; PACAP and VIP were equipotent and 20 times more potent than secretin. On the other hand, the peptides did not affect the glucagon release in the presence of 8.3 mM glucose. 4 On pancreatic vessels, in the range 10−11 to 10−9 m, the three peptides were equipotent in inducing a concentration‐dependent sustained increase in pancreatic flow rate. On the other hand, at the high concentration of 10−7 m PACAP but not VIP provoked a transient decrease of flow rate. 5 This study provides evidence for PACAP/VIP type II receptors mediating insulin and glucagon secretion as well as vasodilatation in rat pancreas. In addition, the different efficacies of secretin suggest that these effects are mediated by different PACAP/VIP type II receptor subtypes.

Effects of phosphate-modified adenine nucleotide analogues on insulin secretion from perfused rat pancreas.

1 The effects of three methylene analogues of adenosine 5′‐triphosphate (ATP) or 5′‐diphosphate (ADP) have been studied on insulin secretion from the isolated perfused pancreas of the rat: 5′‐adenylmethylene diposphonate or β, γ‐methylene ATP, adenosine 5′‐α, β‐methylene triphosphate or α, β‐methylene ATP and adenosine 5′‐α, β‐methylene diphosphonate or α, β‐methylene ADP. 2 β.γ‐Methylene ATP did not elicit any increase of insulin release; α, β‐methylene ATP and α, β‐methylene ADP induced a biphasic stimulation of insulin secretion; this effect was dose‐related between 1.65 and 165 μmol/1. Relative potency ATP/α, β‐methylene ATP was 1.2 and ATP/α, β‐methylene ADP was 0.31. 3 Our results point to the importance of the steric and electronic characteristics of the polyphosphate chain of the analogues of ATP and ADP in inducing an insulin secretory effect. They support the hypothesis of a purine receptor for ATP and ADP.

Evidence for an inhibitory A1 subtype adenosine receptor on pancreatic insulin-secreting cells

The effects of L- and D-phenylisopropyladenosine (L- and D-PIA) were studied on glucose-induced insulin secretion from the isolated perfused rat pancreas. L-PIA at the low dose of 16.5 nM inhibited insulin secretion by 50%. In contrast, D-PIA at 16.5 and 82.5 nM was ineffective. D-PIA used at a 100-fold higher concentration (1.65 microM) than L-PIA induced a similar inhibition of insulin secretion. The inhibitory effect of L-PIA was abolished by 8-phenyltheophylline (1 microM), a potent P1 purinoceptor antagonist. The present experiments provide evidence for an adenosine receptor of the A1 subtype on the insulin-secreting pancreatic cell of rats.

Stimulation of insulin secretion and improvement of glucose tolerance in rat and dog by the P2y‐purinoceptor agonist, adenosine‐5′‐O‐(2‐thiodiphosphate)

1 In vivo effect of a P2y‐purinoceptor agonist, adenosine‐5′‐O‐(2‐thiodiphosphate) (ADPβS), on insulin secretion and glycaemia were studied both in rats and dogs. 2 In anaesthetized rats, i.v. administered ADPβS (0.2 mg kg−1) produced an insulin response dependent on the nutritional state of the animals, since we observed only a transient increase in overnight‐fasted rats and a sustained insulin secretion followed by a reduction in plasma glucose levels in fed rats. During an i.v. glucose tolerance test, ADPβS enhanced insulin release and thus increased the glucose disappearance rate. 3 In anaesthetized fasted dogs, i.v. administered ADPβS (0.1 mg kg−1) increased pancreaticoduodenal insulin output and slightly decreased blood glucose levels. 4 In conscious fasted dogs, orally administered ADPβS (0.1 mg kg−1) transiently increased insulinemia and punctually reduced glycaemia. Furthermore, during an oral glucose tolerance test, orally administered ADPβS at the same dose markedly enhanced insulin secretion and consequently reduced the hyperglycaemia. 5 In conclusion, the P2y‐agonist, ADPβS, is a potent insulin secretagogue in vivo, improves glucose tolerance and is effective after oral administration. Thus, the P2y‐purinoceptors of the β cell may be a target for new antidiabetic drugs.

Evidence for an A2-subtype adenosine receptor on pancreatic glucagon secreting cells.

1 The effects of a 5′‐substituted analogue of adenosine, 5′‐N‐ethylcarboxamidoadenosine (NECA) have been studied on glucagon secretion in vitro, using the isolated pancreas of the rat perfused in the presence of glucose (2.8 mM). 2 NECA provoked a peak of glucagon secretion, the kinetics of which were comparable to those previously obtained with adenosine. The effect was concentration‐dependent and appeared at nanomolar concentrations. The EC50 was approximately 4 × 10−8 M. 3 A comparison of relative potency between adenosine and NECA showed that NECA was about 800 fold more potent than adenosine in inducing glucagon secretion. 4 Theophylline (50 μM) considerably decreased the peak of glucagon secretion induced by 1.65 μM NECA and totally suppressed the effect of 16.5 nM NECA. These results indicate the involvement of an adenosine receptor. 5 These and other previous results (low stereoselectivity of N6‐phenylisopropyladenosine) provide evidence for an adenosine receptor of the A2‐subtype being involved in glucagon secretion.