Internalisation and multiple phosphorylation of γ-Conglutin, the lupin seed glycaemia-lowering protein, in HepG2 cells.

Lupin seed γ-Conglutin is a protein capable of reducing glycaemia in mammalians and increasing glucose uptake by model cells. This work investigated whether γ-Conglutin is internalised into the target cells and undergoes any covalent change during the process, as a first step to understanding its mechanism of action. To this purpose, γ-Conglutin-treated and untreated HepG2 cells were submitted to confocal and transmission electron microscopy. Immune-revelation of γ-Conglutin at various intervals revealed its accumulation inside the cytosol. In parallel, 2D-electrophoresis of the cell lysates and antibody reaction of the blotted maps showed the presence of the protein intact subunits inside the treated cells, whilest no trace of the protein was found in the control cells. However, γ-Conglutin-related spots with an unexpectedly low pI were also observed in the maps. These spots were excised, trypsin-treated and submitted to MS/MS spectrometric analysis. The presence of phosphorylated amino acids was detected. These findings, by showing that γ-Conglutin is internalised by HepG2 cells in an intact form and is modified by multiple phosphorylation, open the way to the understanding of the lupin γ-Conglutin insulin-mimetic activity.

Lupin seed γ-conglutin lowers blood glucose in hyperglycaemic rats and increases glucose consumption of HepG2 cells.

The aim of the present study was to evaluate the effect of a chronic oral γ-conglutin treatment in male Sprague-Dawley rats in which hyperglycaemia had been induced by supplying 10 % d-glucose in drinking-water. A γ-conglutin dosage of 28 mg/kg body weight was daily administered to animals for 21 d. Plasma glucose, insulin and glucose overloading were monitored. Chronic administration of glucose resulted in a statistically significant (P < 0·05) increase in fasting blood glucose (2·5-fold) and insulin (2·7-fold) v. the values recorded in control rats. Simultaneous treatment with γ-conglutin attenuated the rise in plasma glucose (1·9-fold) and insulin (1·8-fold) levels in the glucose-fed rats (P < 0·05). Fasting insulin and homeostasis model of insulin resistance were decreased by 34 and 48 % (P < 0·05), respectively, in the γ-conglutin-treated rats v. the values found in pair-fed animals. To confirm these results with a different approach, HepG2 cells, grown for 24 and 48 h in Dulbecco's minimum essential medium containing different glucose concentrations (5·5, 11·1 and 16·5 mmol/l), were exposed to 10 µmol/l γ-conglutin with or without 10 mmol/l metformin or 100 nmol/l insulin. γ-Conglutin increased glucose consumption (from 1·5- to 2·5-fold) in HepG2 cells, under all experimental conditions; this effect was more evident after 48 h incubation. Moreover, in this in vitro model, the addition of γ-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 γ-conglutin in the control of glycaemia.

Heterologous expression and purification of the soybean 7S globulin α' subunit extension region: in vitro evidence of its involvement in cell cholesterol homeostasis.

In a previous paper, the biological activity of a 216-amino acid recombinant truncated form of the soybean 7S globulin α' subunit, known to control cholesterol and triglyceride homeostasis, was described. In this work, a shorter version of the polypeptide chain, spanning 142 amino acid residues from the N-terminus and thus exclusively including the so-called extension region, was cloned and overexpressed in Pichia pastoris. The yield of the recombinant polypeptide, which was termed α'E, was 8-fold greater than the previous truncated version. The α'E polypeptide was purified by simple conventional biochemical techniques to make it available for biological assays. Human hepatoma cell lines (Hep G2) were used to monitor the uptake and degradation of labeled low-density lipoproteins (LDL), according to an established procedure. The LDL uptake (+86%) and degradation (+94%) by cells tested at the highest α'E dose (2 µM) were similar to those found in cells incubated with 1 µM simvastatin, a potent inhibitor of cholesterol biosynthesis. Additionally, the cell response to α'E was found to be dose-dependent. The present findings strongly suggest that this recombinant polypeptide, or a fragment thereof, is the molecular determinant for cholesterol homeostasis and open new prospects for understanding the mechanism involved in this biological response, as a gateway to its utilization in lipid-lowering therapies.

Cloning, yeast expression, purification and biological activity of a truncated form of the soybean 7S globulin alpha' subunit involved in Hep G2 cell cholesterol homeostasis.

A truncated form of alpha' chain (talpha'), the soybean 7S globulin subunit previously demonstrated to be active in controlling the cholesterol and triglyceride homeostasis in in vitro and in vivo models, was cloned and expressed in the yeast Pichia pastoris. The recombinant polypeptide spanned 216 amino acid residues from the N-terminal side and included the N-terminal extension region of the soybean subunit. The talpha' polypeptide was purified by conventional biochemical techniques, and its potential to modulate the activity of low-density lipoprotein (LDL) receptor was evaluated in a human hepatoma cell line (Hep G2) by monitoring the uptake and degradation of labeled LDL. The LDL uptake (+192%) and degradation (+143%) by cells tested at the highest talpha' dose (8 microM) were similar to those found in cells incubated with 1 microM simvastatin, a potent inhibitor of cholesterol biosynthesis. The cell response to talpha' was found to be dose dependent. The use of a recombinant polypeptide ruled out the involvement of any other soybean component. These findings open new prospects in the studies aimed at identifying soybean regulatory (poly)peptide(s) and the mechanism involved in this biological response, as a gateway to their utilization for the management of human health.

Fatty acid profiles of blood lipids in a population group in Tibet: correlations with diet and environmental conditions.

The aim of this study was to compare blood fatty acid profiles of two population groups: Italian and Tibetan, differing with regard to ethnic, life style and environmental aspects. Additionally the collection of two staple foods provided the opportunity to analyze typical Tibetan dishes. A new, simple, rapid, and substantially non invasive method for fatty acid (FA) analysis of blood lipids was applied to healthy Italian (n=14) and Tibetan (n=13) subjects. Blood drops obtained from the ear lobe of Tibetans or the fingertip of Italians were adsorbed by a special strip of paper and processed for fatty acid analysis. The fatty acid profiles of the two groups are different, and environmental factors, such as dietary fats and altitudes of Milan, Italy (a low altitude site), and Lhasa, Tibet (a high altitude site) appear to contribute to these differences. More specifically, in Ti-betans higher levels of monounsaturated fatty acids, including the 22 and 24 carbon molecules, were found. This appears to be derived mainly from locally consumed fats (mustard seed oil), and are associated with lower levels of total polyunsaturated fatty acids and higher levels of selected omega 3 fatty acids, when compared to the Italians. These relatively higher levels of monounsaturated fatty acids may also indicate means of adaptation to local prooxidant conditions. The observed differences in blood fatty acid profiles in Tibetans vs. Italians appear to result both from dietary factors and adaptation to local environmental conditions such as the high altitude of the Tibetan location.

Proteins of white lupin seed, a naturally isoflavone-poor legume, reduce cholesterolemia in rats and increase LDL receptor activity in HepG2 cells.

White lupin (Lupinus albus, L.), a widely cultivated crop that has been consumed for many years in Western Europe, may provide a useful alternative for individuals wishing to substitute animal with plant proteins for cardiovascular disease prevention. Lupin seeds have a very low content of isoflavones, and lupin protein isolates are essentially isoflavone free. In rats fed a casein-based cholesterol + cholic acid diet, a relatively low daily intake (50 mg/d by gavage for 2 wk) of total lupin protein extract reduced plasma total and VLDL + LDL cholesterol concentrations by 21 and 30%, respectively (both P<0.001). In an attempt to elucidate the lipid-lowering mechanism, LDL receptor activity was evaluated in a human hepatoma cell line (HepG2). In this model, the lupin total protein extract was essentially inactive, whereas one purified minor protein component, conglutin gamma, had a remarkable upregulatory effect, with maximal increases of 53 and 21% (both P<0.05) for LDL uptake and degradation, respectively. This initial study indicates that lupin, although isoflavone free, has hypocholesterolemic activity similar to that of other leguminous proteins in an established animal model. Further, the cholesterol reduction appears to be associated with stimulation of LDL receptors by a well-defined protein component of the lupin seeds as demonstrated by in vitro studies.

Soy proteins reduce progression of a focal lesion and lipoprotein oxidiability in rabbits fed a cholesterol-rich diet.

The effects of different dietary proteins on the progression of a focal atheromatous lesion and on lipoprotein oxidiability were studied in male New Zealand rabbits. Focal lesions were induced on common carotid arteries by applying an electric current, using a bipolar microcoagulator. After surgery, animals were fed for 90 days two different diets, both with 1% cholesterol, 15% saturated fatty acids and 20% protein: the protein source was constituted in one group (SOY) by 16% soy protein isolate plus 4% milk whey proteins, in the other (CASEIN) by 16% casein plus 4% milk whey proteins. Lower levels of plasma cholesterol and triglycerides (-47 and -65%, respectively) (P < 0.05) were detected in the SOY versus the CASEIN group at the end of treatment. Cryosection analyses of the carotids, indicated a highly significant reduction (-39%; P < 0.05) in the focal lesion progression in the SOY versus the CASEIN group. Copper-mediated oxidation of low-density lipoprotein (LDL) from rabbits fed the two different diets, performed in vitro by analysis of conjugated diene formation, showed a significantly longer lag phase in the SOY (150 +/- 5 min) versus the CASEIN animals (20 +/- 3 min) (P < 0.05). These data, while confirming the well-known lipid lowering properties of soy proteins, indicate, in this animal model, a remarkable activity on a focal atheromatous lesion, possibly also linked to a powerful antioxidant activity.

Subcellular localization of soybean 7S globulin in HepG2 cells and LDL receptor up-regulation by its alpha' constituent subunit.

The aims of this work were to monitor the subcellular localization of soybean 7S globulin in HepG2 cells and determine its interaction with cell protein components, by using laser-induced fluorescence capillary electrophoresis (LIF-CE). Furthermore, we evaluated in the same cell line the involvement of the alpha' constituent subunit from 7S globulin in the modulation of LDL catabolism. The results indicated a main fluorescein isothiocyanate-tagged 7S globulin (FITC-7S) component in the cytosolic fraction, that was not present in the nuclear compartment. The electrophoretic mobility of this tagged component suggested either a dissociation of the 7S oligomer or its partial intracellular degradation. Interactions of soybean 7S globulin with FITC-thioredoxin 1 and FITC-cyclophilin B, HepG2 cell membrane proteins, were demonstrated in in vitro assays. In a separate experiment with HepG2 cells, the ability of the alpha' subunit purified from soybean 7S globulin to modulate the activity of the LDL receptors was evaluated by tracking the uptake and degradation of labeled LDL. The up-regulation of LDL receptors by the alpha' subunit, as further confirmed by a LDL receptor promoter assay, was significantly greater than that found in the control cells. In conclusion, this study, while confirming our previous indirect evidence of the key role of alpha' subunit on the cell cholesterol homeostasis, reveals a potentially interesting association of soybean 7S globulin with proteins, such as thioredoxin 1 and cyclophilin B, that are involved in cell protection against oxidative stress.

Monitoring of minimal residual disease after CHOP and rituximab in previously untreated patients with follicular lymphoma.

Minimal residual disease (MRD) following sequential administration of CHOP and rituximab was studied in previously untreated patients with follicular lymphoma. At diagnosis, the presence of Bcl-2/IgH-positive cells in the peripheral blood (PB) and/or bone marrow (BM) was demonstrated in all patients (n = 128) by polymerase chain reaction (PCR) analysis. Patients who achieved a clinical response following CHOP but remained PCR-positive were eligible for rituximab (375 mg/m(2) intravenously, weekly for 4 weeks). After CHOP, 57% achieved a complete response (CR), 37% a partial response (PR), and 6% were nonresponders (NR). At this stage, patients proving PCR-negative (n = 41) or failing to achieve a clinical response (n = 8) were excluded from rituximab treatment. Seventy-seven patients received rituximab and entered a scheduled MRD follow-up program. At the first molecular follow-up (+12 weeks), 59% had converted to PCR negativity in the BM and PB, with a further increase documented at the second control (+28 weeks) with 74% PCR negative. At the last molecular follow-up (+44 weeks), 63% of the patients remained PCR negative. At 3 years, the estimated overall survival of all patients is 95% (95% confidence interval [CI], 86-98). For patients achieving PCR-negative status following CHOP and therefore excluded from rituximab treatment, freedom from recurrence (FFR) was 52% (95% CI, 28-71). For patients treated with rituximab, a durable PCR-negative status was associated with a better clinical outcome since FFR was 57% (95% CI, 23-81) compared with 20% (95% CI, 4-46) in patients who never achieved or lost the molecular negativity (P <.001).