Introduction
Chronic non-communicable diseases such as diabetes are increasing public health problems worldwide.The
prevalence of these diseases will increase drastically in the coming years particularly in developing countries.
Diabetes is expected to affect approximately 300 million people by 2025.
Hypoglycemic effect of cooked lupinus mutabilis and its purified alkaloids in subjects with type-2 diabetes, 2012
There is a constant urge to develop new therapies with better effects, lower side effects at lower prices
to treat these diseases. Lupinus species and their derivates are good candidates to be used as hypoglycaemic agents.
Hypoglycemic effect of Lupinus mutabilis in healthy volunteers and subjects with dysglycemia, 2012
Lupin is a leguminous plant which produces edible grains. Lupin seeds have a peculiar composition: rich in proteins, fibres and oil, no starch and only few other carbohydrates. These main components are extremely valuable and make lupin an important crop for human nutrition. Some lupin proteins display remarkable nutraceutical activities, such as the plasma glucose-lowering γ-conglutin.
Lupin: classification and geographic distribution
Lupinus, commonly known as lupin or lupine (North America), is a genus of flowering plants in the legume family (Fabaceae). The genus comprises about 280 species (Hughes), with major centers of diversity in South and Western North America (Subgen. Platycarpos (Wats.) Kurl.), parts of the Southern Hemisphere (New Zealand and parts of Australia) and the Andes and secondary centers in the Mediterranean region and Africa (Subgen. Lupinus)
Lupin classification; page 5
Geographic distribution of lupin; page 6
Lupinus albus, commonly known as the white lupin, is a member of the genus Lupinus in the family Fabaceae. It is a traditional pulse cultivated in the Mediterranean region.
The white lupin is annual, more or less pubescent plant, 30 to 120 cm high, has a wide distribution in the Mediterranean region. White Lupine is widely spread as wild plants throughout the southern Balkans, the islands of Sicily, Corsica and Sardinia, and the Aegean Sea, as well as in Palestine and western Turkey. Occurs in meadows, pastures, and grassy slopes, predominantly on sandy and acid soils. It is cultivated over all the Mediterranean region and also in Egypt, Sudan, Ethiopia, Syria, Central and Western Europe, USA and South America, Tropical and Southern Africa, Russia, and Ukraine. The ancient culture of white lupin under the local name "hanchcoly" was practiced until recently in Western Georgia.
White lupin is distinct within the vast and polymorphous genus Lupinus L. for small variation of morphological characters. However, it has wide intraspecific variability in physiological plant properties: duration of vernalization time and growth rate, photoperiodic sensitivity, shape tolerance, drought resistance, cold- and winter-hardiness. There are winter and spring forms of white lupin. Duration of growing period under spring sowing varies from 106 to 180 days, seed mass per plant changes from 2.2 to 40 g, green mass yield per from 9 to 250 g, protein content in seed from 35.0 to 53.7%, and oil content from 6.2 to 12.0%.
Lupin seed is referred to as an antidiabetic product in traditional medicine, since conglutin-g, a lupin seed glycoprotein, was found to cause a significant plasma glucose reduction when orally administered to rats in glucose overload trials. Conglutin-g was identified as being responsible for the claimed biological activity
Insulin-mimetic action of conglutin-γ, a lupin seed protein, in mouse myoblasts, 2011
Legume seeds also contain a number of biologically active proteins that play many specialized roles.
Conglutin gamma, a lupin seed protein, binds insulin in vitro and reduces plasma glucose levels of hyperglycemic rats, 2004
Discussion and Molecular mechanism
The anti-diabetic activity of toasted lupin seeds was first described, in the middle of the last century, by Ferranini & Pirolli and by Orestano, who proposed lupin as a substitute for the insulin therapy in mild-to-medium diabetes mellitus, but no further studies have been carried out to identify the molecule responsible of this biological effect.
Lovati et al. study provides the in vivo and in vitro evidence of the involvement of g-conglutin on cell glucose homeostasis, thus suggesting the potential use of this food protein in the control of glycaemia in patients with manifest or pre-clinical diabetes as well as for applications as functional foods and dietary supplements.
Conglutin g is a glycoprotein of Mr relative molecular mass around 47 kDa. It is composed of two disulphide bridged subunits of 29 and 17 kDa.Conglutin g displays unique properties of its own, since its amino acid sequence does not match any other legume protein canonical sequence: it binds divalent metal ions, especially Zn++ and Ni++ , it is not cleaved during seed germination and it is also unusually resistant to in vitro proteolysis, unless fully denatured .
Conglutin gamma, a lupin seed protein, binds insulin in vitro and reduces plasma glucose levels of hyperglycemic rats, 2004
Conglutin-g in its native conformation is unusually resistant to proteolysis by trypsin
Insulin-mimetic action of conglutin-γ, a lupin seed protein, in mouse myoblasts, 2011
γ-conglutin electrophoresis:
Combined 2-D electrophoretic approaches for the study of white lupin mature seed storage proteome
Predictive 3D homology model of γ-conglutin:
Disulphide bridges are indicated in yellow, whereas the two loops involved in the interaction with the enzyme are in pink and red, respectively. A detail of the structural model of the xylanase–TAXI complex (PDB code: 1T6G) corresponding to the enzyme–inhibitor interaction region, is reported in the inset. The relevant region of the endo-glucanase enzyme is reported as a green strand. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
In the experimental conditions used (detailed in the Methods and materials section), the effect of the oral administration of conglutin g to rats under glucose overloading produced a significant reduction of plasma glucose with respect to the control. This effect was greatest at 30 minutes from the glucose overloading and persisted until 60 minutes. After 90 minutes, glucose levels were not statistically different from the control group. The observed effect appeared to be dose-dependent: in particular, the effect of the highest conglutin g dose did not differ statistically from that measured with metformin, a wellknown glucose lowering drug.
The present findings, by showing a specific interaction of conglutin g with insulin and the effects on the animal models, candidate this protein as the molecule responsible for the regulatory effect of lupin seeds on glycemia.
Conglutin gamma, a lupin seed protein, binds insulin in vitro and reduces plasma glucose levels of hyperglycemic rats, 2004
Influence of γ-conglutin on plasma glucose concentrations of rats during glucose overloading trials. Plasma glucose was assayed in rats at various times from glucose overload (control: continuous line) upon pretreatment with 30 ("dash-dot-dot" line), 60 ("dash-dot" line), 120 (broken line) mg/kg body weight of γ-conglutin or 50 mg/ KG body weight metformin (dotted line). Each value represents the mean of five rats. Vertical bars indicate standard error of each mean value.
Conglutin gamma, a lupin seed protein, binds insulin in vitro and reduces plasma glucose levels of hyperglycemic rats, 2004
Insulin is responsible for proteosynthesis control through IRS/AKT/P70S6k/PHAS1 pathways modulation, glucose homeostasis through PKC/Flotillin-2/caveolin-3/Cbl activation and muscle differentiation/hypertrophy via muscle-specific MHC gene transcription control.
Conglutin-g may regulate muscle energy metabolism, protein synthesis and MHC gene transcription through the modulation of the same insulin signal ling pathway,suggesting the potential therapeutic use of this natural legumeprotein in the treatment of diabetes and other insulin-resistant conditions, aswell as the potential conglutin-g influence onmuscle cells differentiation and regulation of muscle growth.
Conglutin-g was identified as the responsible molecule for the claimed anti-diabetic properties of lupin seeds in traditional medicine. Since insulin-binding to its own receptor causes a series of phosphorylation/dephosphorylation reactions, which lead the insulin signal from the receptor to the final metabolic and myogenic pathways, we firstly hypothesize that the effect of conglutin-g on blood glucose is due to an insulin-mimetic effect of the protein at the level of the intracellular pathway insulin receptor/IRS-1/PI-3-kinase, eventually leading to the recruitment and translocation of GLUT4. Secondly, as insulin promotes muscle anabolism, we hypothesize the activation of a protein synthetic and myogenic process by conglutin-g. The aim of this work was to test the effect of conglutin-g in an in vitro model of mouse myoblasts, assessing the modulation of muscle-specific genes and the phosphorylation/activation of intracellular kinases involved in the insulin signalling cascade. Our results indicate that conglutin-g shares, with insulin, common effects on the intracellular kinases tested in this work, suggesting a possible therapeutic indication as an insulin-mimetic agent.
Intracellular pathways of insulin signaling. Ins: insulin; IRS-1: insulin Receptor Substrate 1; PI3K: phosphatidylinositol 3-kinase; AKT also called PKB: protein kinase B; p70S6K: p70 ribosomal protein S6 kinase; PHAS1 also called 4EBP1: eukaryotic initiation factor 4E-binding protein-1; eIF4E/4G/4A: eukaryotic initiation factor13 4E/4G/4A; PKC: protein kinase C; Flo-2: flotillin-2; Glut4: glucose transporter 4; Cav-3: caveolin-3; CBL: tyrosine phosphorylated protein of the protooncogenes c-Cbl and Cbl-b; CAP: Cbl associated protein; ERK1/2: extracellular signal-regulated kinases 1/2.
Insulin-mimetic action of conglutin-γ, a lupin seed protein, in mouse myoblasts, 2011
These previous findings need to be confirmed in in vivo models of pre-diabetic or diabetic state in order to assess g-conglutin actual potential. On this basis, the aim of the present study was to evaluate the effect of a chronic oral administration of g-conglutin in male Sprague–Dawley rats in which hyperglycaemia was induced by supplying 10% D-glucose in drinking-water(15) in addition to a normal chow diet during 3 weeks. In this animal model, chronic glucose feeding resulted in non-insulin-dependent diabetes as reflected by the increase in both blood glucose and insulin levels.In addition to these trials, further experiments were carried out on HepG2 cells, 2012, a human hepatoma cell line, to assess g-conglutin effects on glucose consumption and to get hints on the possible mechanism.
In Lovati et al. study, the 10% glucose supplied in drinking water induced hyperglycaemia and hyperinsulinaemia in rats similar to that observed in human subjects. No side effects have been detected during the experimental period, such as those recorded in animals undergoing streptozotocin or alloxan treatment to mimic diabetes. g-Conglutin administration has been demonstrated to counteract the plasma glucose increase as well as to improve the insulin sensitivity, normally reduced by the glucose-rich drinking-water. In the g-conglutin-treated rats, the insulin sensitisation was increased significantly, as indicated by the 48% reduction in the homeostasis model of IR. It is worth noting that the hypoglycaemic effect in vivo was obtained by the use of a preparation, which contained a g-conglutin amount corresponding to the lowest dose previously used in acute trials of glucose overload. Moreover, lower glucose levels were detected in g-conglutin-treated rats following oral glucose overload; these results were confirmed by lower glycaemia in fasting and 2 h postprandial conditions. The mechanism/s, underlying the present results, are currently under investigation, but the hypothesis that g-conglutin could act as an insulin-like agent should not be excluded.
g-Conglutin increased glucose consumption (from 1.5 to 2.5 fold) in HepG2 cells, 2012, under all experimental conditions; this effect was more evident after 48 h incubation. Moreover, in this in vitro model, the addition of g-conglutin potentiated the activity of insulin and metformin in cell glucose consumption. These findings extend the previous ones and suggest the potential use of lupin g-conglutin in the control of glycaemia.
In conclusion, the Lovati et al. study provides the in vivo and in vitro evidence of the involvement of g-conglutin on cell glucose homeostasis, thus suggesting the potential use of this food protein in the control of glycaemia in patients with manifest or pre-clinical diabetes as well as for applications as functional foods and dietary supplements.
Lupin seed γ-conglutin lowers blood glucose in hyperglycaemic rats and increases glucose consumption of HepG2 cells, 2012
Consumption of cooked L. mutabilis or its purified alkaloids decreased blood glucose and insulin levels. The decreases in serum glucose concentrations from base line to 90 minutes were statistically significant within both treatment groups; however, there were not differences between groups. Serum insulin levels were also decreased in both groups however the differences were not statistically significant. None of the volunteers in either group presented side effects.
Hypoglycemic effect of cooked lupinus mutabilis and its purified alkaloids in subjects with type-2 diabetes, 2012
In the Fornasini et al. study, a phase II clinical trial was conducted to assess the role of raw Lupinus mutabilis on blood glucose and insulin in normoglycemic and dysglycemic subjects. Results show that consumption of L. mutabilis by normal weight healthy young individuals did not change importantly blood glucose and insulin levels. On the other hand, consumption of similar doses of lupinus by dysglycemic individuals (fasting glucose > 100 mg/dL) decreased significantly blood glucose. (FIG.1) Lupinus effects were greater in those subjects with higher basal glucose levels. Glucose lowering effects of lupinus were not observed after soy intake that was used as control. A statistically significant reduction in insulin levels was also observed in the lupinus group compared with the soy group after 60 minutes of treatment. Furthermore, only treatment with lupines improved insulin resistance in dysglycemic subjects. These data demonstrate that lupinus consumption could be a feasible and low cost alternative to treat chronic hyperglycemic diseases.
Hypoglycemic effect of Lupinus mutabilis in healthy volunteers and subjects with dysglycemia, 2012
