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4-Hydroxyisoleucine, a peculiar amino acid extracted from fenugreek seeds and never found in mammalian tissues, exhibits interesting insulinotropic activity. To investigate the structural requirements for this stimulating effect, the insulinotropic activity of the major isomer (2S,3R,4S) of 4-hydroxyisoleucine, in the presence of 8. 3 mM glucose, was compared to that of (1) its minor isomer (2R,3R, 4S) (2) its lactone form, (3) classical structurally related amino acids, and (4) synthetic monomethylated analogues. In the isolated, ex vivo, perfused rat pancreas, only the major isomer of 4-hydroxyisoleucine (200 microM) potentiated insulin release. On incubated isolated rat islets, the threshold concentration for a significant increase (P<0.05) in insulin release was 200 microM for (2S,3R,4S) 4-hydroxyisoleucine, 500 microM for (2S,4R) and (2S,4S) gamma-hydroxynorvalines as well as (2S,3S) and (2S,3R) gamma-hydroxyvalines, and 1 mM or more for other congeners. In conclusion, the insulinotropic properties of 4-hydroxyisoleucine, in the micromolar range, are seen only in the presence of the linear major isoform; they also require carbon alpha in S-configuration, full methylation and carbon gamma-hydroxylation.

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We analyzed the production of reactive oxygen species, the accumulation of salicylic acid (SA), and peroxidase activity during the incompatible interaction between cotyledons of the cotton (Gossypium hirsutum) cv Reba B50/Xanthomonas campestris pv malvacearum (Xcm) race 18. SA was detected in petioles of cotyledons 6 h after infection and 24 h post inoculation in cotyledons and untreated leaves. The first peak of SA occurred 3 h after generation of superoxide (O(2)(.-)), and was inhibited by infiltration of catalase. Peroxidase activity and accumulation of SA increased in petioles of cotyledons and leaves following H(2)O(2) infiltration of cotyledons from 0.85 to 1 mM. Infiltration of 2 mM SA increased peroxidase activity in treated cotyledons and in the first leaves, but most of the infiltrated SA was rapidly conjugated within the cotyledons. When increasing concentrations of SA were infiltrated 2. 5 h post inoculation at the beginning of the oxidative burst, the activity of the apoplastic cationic O(2)(.-)-generating peroxidase decreased in a dose-dependent manner. We have shown that during the cotton hypersensitive response to Xcm, H(2)O(2) is required for local and systemic accumulation of SA, which may locally control the generation of O(2)(.-). Detaching cotyledons at intervals after inoculation demonstrated that the signal leading to systemic accumulation of SA was emitted around 3 h post inoculation, and was associated with the oxidative burst. SA produced 6 h post infection at HR sites was not the primary mobile signal diffusing systemically from infected cotyledons.

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We report the characterization of a new insulinotropic compound, 4-hydroxyisoleucine. This amino acid has been extracted and purified from fenugreek seeds, which are known in traditional medicine for their antidiabetic properties. 4-Hydroxyisoleucine increases glucose-induced insulin release, in the concentration range of 100 micromol/l to 1 mmol/l, through a direct effect on isolated islets of Langerhans from both rats and humans. The stimulating effect of 4-hydroxyisoleucine was strictly glucose dependent; indeed, ineffective at low (3 mmol/l) or basal (5 mmol/l) glucose concentrations, the amino acid potentiated the insulin secretion induced by supranormal (6.6-16.7 mmol/l) concentrations of glucose. In addition, in the isolated perfused rat pancreas, we could show 1) that the pattern of insulin secretion induced by 4-hydroxyisoleucine was biphasic, 2) that this effect occurred in the absence of any change in pancreatic alpha- and delta-cell activity, and 3) that the more glucose concentration was increased, the more insulin response was amplified. Moreover, 4-hydroxyisoleucine did not interact with other agonists of insulin secretion (leucine, arginine, tolbutamide, glyceraldehyde). Therefore, we conclude that 4-hydroxyisoleucine insulinotropic activity might, at least in part, account for fenugreek seeds' antidiabetic properties. This secretagogue may be considered as a novel drug with potential interest for the treatment of NIDDM.

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The seeds of fenugreek (Trigonella foenum graecum L.) are traditionally assumed to have restorative properties. We have recently shown that a fenugreek seed extract containing steroid saponins increased food consumption and induced hypocholesterolemia in rats. This study aims to investigate the specific role of purified steroid saponins in these properties. For this purpose, an original technique for extraction and purification of steroid saponins was carried out. Thereafter, the effects of these steroid saponins were investigated on feeding behavior and metabolic endocrine changes in normal and diabetic rats. All the steroid saponins (furostanol type) were extracted from the seeds and separated from all other constituents of the entire extract by using several purification procedures to give an extract containing at least 90% of steroid saponins. Pharmcological experiments were performed in vivo in normal and streptozotocin diabetic rats: steroid saponins were administered chronically mixed with food (12.5 mg/day per 300 g body weight). Our data show that the treatment with steroid saponins significantly increased food intake and the motivation to eat in normal rats, while modifying the circadian rhythm of feeding behavior; it also stabilized the food consumption in diabetic rats, which resulted in a progressive weight gain in these animals, in contrast to untreated diabetic controls. Both in normal and diabetic rats, steroid saponins decreased total plasma cholesterol without any change in triglycerides. In conclusion, the present work reports a clear methodology to obtain all the steroid saponins and demonstrates that these saponins enhance food consumption and motivation to eat, and reduce plasma cholesterol levels in rats.

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