Evaluation of the antidiabetic activity of DPP IV resistant N-terminally modified versus mid-chain acylated analogues of glucose-dependent insulinotropic polypeptide

Glucose dependent insulinotropic polypeptide (GIP) is a gastrointestinal hormone with therapeutic potential for type 2 diabetes due to its insulin-releasing and antihyperglycaemic actions. However, development of GIP-based therapies is limited by N-terminal degradation by DPP IV resulting in a very short circulating half-life. Numerous GIP analogues have now been generated exhibiting DPP IV resistance and extended bioactivity profiles. In this study, we report a direct comparison of the long-term antidiabetic actions of three such GIP molecules, N-AcGIP, GIP(LyS(37)PAL) and N-AcGIP(LyS(37)PAL) in obese diabetic (ob/ob) mice. An extended duration of action of each GIP analogue was demonstrated prior to examining the effects of once daily injections (25 nmol kg(-1) body weight) over a 14-day period. Administration of either N-AcGIP, GIP(LyS(37)PAL) or N-AcGIP(LyS37PAL) significantly decreased non-fasting plasma glucose and improved glucose tolerance compared to saline treated controls. All three analogues significantly enhanced glucose and nutrient-induced insulin release, and improved insulin sensitivity. The metabolic and insulin secretory responses to native GIP were also enhanced in 14-day analogue treated mice, revealing no evidence of GIP-receptor desensitization. These effects were accompanied by significantly enhanced pancreatic insulin following N-AcGIP(Lys(37)PAL) and increased islet number and islet size in all three groups. Body weight, food intake and circulating glucagon were unchanged. These data demonstrate the therapeutic potential of once daily injection of enzyme resistant GIP analogues and indicate that N-AcGIP is equally as effective as related palmitate derivatised analogues of GIP. (c) 2006 Elsevier Inc. All rights reserved.

A novel, long-acting agonist of glucose-dependent insulinotropic polypeptide suitable for once-daily administration in type 2 diabetes

Glucose-dependent insulinotropic polypeptide (GIP) is a gastrointestinal hormone with a potentially therapeutic role in type 2 diabetes. Rapid degradation by dipeptidylpeptidase IV has prompted the development of enzyme-resistant N-terminally modified analogs, but renal clearance still limits in vivo bioactivity. In this study, we report long-term antidiabetic effects of a novel, N-terminally protected, fatty acid-derivatized analog of GIP, N-AcGIP(LysPAL(37)), in obese diabetic (ob/ob) mice. Once-daily injections of N-AcGIP(LysPAL(37)) over a 14-day period significantly decreased plasma glucose, glycated hemoglobin, and improved glucose tolerance compared with ob/ob mice treated with saline or native GIP. Plasma insulin and pancreatic insulin content were significantly increased by N-AcGIP(LysPAL(37)). This was accompanied by a significant enhancement in the insulin response to glucose together with a notable improvement of insulin sensitivity. No evidence was found for GIP receptor desensitization and the metabolic effects of NAcGIP(LysPAL(37)) were independent of any change in feeding or body weight. Similar daily injections of native GIP did not affect any of the parameters measured. These data demonstrate the ability of once-daily injections of N-terminally modified, fatty acid-derivatized analogs of GIP, such as N-AcGIP(LysPAL(37)), to improve diabetes control and to offer a new class of agents for the treatment of type 2 diabetes.

Diabetes-induced activation of protein kinase C inhibits store-operated Ca2+ uptake in rat retinal microvascular smooth muscle.

AIMS/HYPOTHESIS: To assess the effects of diabetes-induced activation of protein kinase C (PKC) on voltage-dependent and voltage-independent Ca2+ influx pathways in retinal microvascular smooth muscle cells. METHODS: Cytosolic Ca2+ was estimated in freshly isolated rat retinal arterioles from streptozotocin-induced diabetic and non-diabetic rats using fura-2 microfluorimetry. Voltage-dependent Ca2+ influx was tested by measuring rises in [Ca2+]i with KCl (100 mmol/l) and store-operated Ca2+ influx was assessed by depleting [Ca2+]i stores with Ca2+ free medium containing 5 micromol/l cyclopiazonic acid over 10 min and subsequently measuring the rate of rise in Ca2+ on adding 2 mmol/l or 10 mmol/l Ca2+ solution. RESULTS: Ca2+ entry through voltage-dependent L-type Ca2+ channels was unaffected by diabetes. In contrast, store-operated Ca2+ influx was attenuated. In microvessels from non-diabetic rats 20 mmol/l D-mannitol had no effect on store-operated Ca2+ influx. Diabetic rats injected daily with insulin had store-operated Ca2+ influx rates similar to non-diabetic control rats. The reduced Ca2+ entry in diabetic microvessels was reversed by 2-h exposure to 100 nmol/l staurosporine, a non-specific PKC antagonist and was mimicked in microvessels from non-diabetic rats by 10-min exposure to the PKC activator phorbol myristate acetate (100 nmol/l). The specific PKCbeta antagonist LY379196 (100 nmol/l) also reversed the poor Ca2+ influx although its action was less efficacious than staurosporine. CONCLUSION/INTERPRETATION: These results show that store-operated Ca2+ influx is inhibited in retinal arterioles from rats having sustained increased blood glucose and that PKCbeta seems to play a role in mediating this effect.

Chemical ablation of gastric inhibitory polypeptide receptor action by daily (Pro3) GIP administration improves glucose tolerance and ameliorates insulin resistance and abnormalities of islet structure in obesity-diabetes.

Glucose-dependent insulinotropic polypeptide (gastric inhibitory polypeptide [GIP]) is an important incretin hormone secreted by endocrine K-cells in response to nutrient ingestion. In this study, we investigated the effects of chemical ablation of GIP receptor (GIP-R) action on aspects of obesity-related diabetes using a stable and specific GIP-R antagonist, (Pro3)GIP. Young adult ob/ob mice received once-daily intraperitoneal injections of saline vehicle or (Pro3)GIP over an 11-day period. Nonfasting plasma glucose levels and the overall glycemic excursion (area under the curve) to a glucose load were significantly reduced (1.6-fold; P <0.05) in (Pro3)GIP-treated mice compared with controls. GIP-R ablation also significantly lowered overall plasma glucose (1.4-fold; P <0.05) and insulin (1.5-fold; P <0.05) responses to feeding. These changes were associated with significantly enhanced (1.6-fold; P <0.05) insulin sensitivity in the (Pro3)GIP-treated group. Daily injection of (Pro3)GIP reduced pancreatic insulin content (1.3-fold; P <0.05) and partially corrected the obesity-related islet hypertrophy and ß-cell hyperplasia of ob/ob mice. These comprehensive beneficial effects of (Pro3)GIP were reversed 9 days after cessation of treatment and were independent of food intake and body weight, which were unchanged. These studies highlight a role for GIP in obesity-related glucose intolerance and emphasize the potential of specific GIP-R antagonists as a new class of drugs for the alleviation of insulin resistance and treatment of type 2 diabetes.

Demonstration of increased concentrations of circulating glycated insulin in human Type 2 diabetes using a novel and specific radioimmunoassay.

Aims/hypothesis: Glycation of insulin, resulting in impaired bioactivity, has been shown within pancreatic beta cells. We have used a novel and specific radioimmunoassay to detect glycated insulin in plasma of Type 2 diabetic subjects.

Methods: Blood samples were collected from 102 Type 2 diabetic patients in three main categories: those with good glycaemic control with a HbA1c less than 7%, moderate glycaemic control (HbA1c 7–9%) and poor glycaemic control (HBA1c greater than 9%). We used 75 age- and sex-matched non-diabetic subjects as controls. Samples were analysed for HbA1c, glucose and plasma concentrations of glycated insulin and insulin.

Results: Glycated insulin was readily detected in control and Type 2 diabetic subjects. The mean circulating concentration of glycated insulin in control subjects was 12.6±0.9 pmol/l (n=75). Glycated insulin in the good, moderate and poorly controlled diabetic groups was increased 2.4-fold (p<0.001, n=44), 2.2- fold (p<0.001, n=41) and 1.1-fold (n=17) corresponding to 29.8±5.4, 27.3±5.7 and 13.5±2.9 pmol/l, respectively.

Conclusion/interpretation: Glycated insulin circulates at noticeably increased concentrations in Type 2 diabetic subjects. [Diabetologia (2003) 46:475–478]

Incretin hormone mimetics and analogues in diabetes therapeutics

The incretin hormones glucagon-like peptide-I (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are physiological gut peptides with insulin-releasing and extrapancreatic glucoregulatory actions. Incretin analogues/mimetics activate GLP-I or GIP receptors whilst avoiding physiological inactivation by dipeptidyl peptidase 4 (DPP-4), and they represent one of the newest classes of antidiabetic drug. The first clinically approved GLP-1 mimetic for the treatment of type-2 diabetes is exenatide (Byetta/exendin) which is administered subcutaneously twice daily. Clinical trials of liraglutide, a GLP-1 analogue suitable for once-daily administration, are ongoing. A number of other incretin molecules are at earlier stages of development. This review discusses the various attributes of GLP-1 and GIP for diabetes treatment and summarises current clinical data. Additionally, it explores the therapeutic possibilities offered by preclinical agents, such as non-peptide GLP-1 mimetics, GLP-1/glucagon hybrid peptides, and specific GIP receptor antagonists.

Characterisation and glucoregulatory actions of a novel acylated form of the (Pro(3))GIP receptor antagonist in type 2 diabetes

In this study, we tested the biological activity of a novel acylated form of (Pro(3))glucose-dependent insulinotropic polypetide [(Pro3)GIP] prepared by conjugating palmitic acid to Lys(16) to enhance its efficacy in vivo by promoting binding to albumin and extending its biological actions. Like the parent molecule (Pro(3))GIP, (Pro(3))GIPLys(16)PAL was completely stable to the actions of DPP-IV and significantly (p <0.01 to p <0.001) inhibited GIP-stimulated cAMP production and cellular insulin secretion. Furthermore, acute administration of (Pro(3))GIPLys(16)PAL also significantly (p <0.05 to p <0.001) countered the glucose-lowering and insulin-releasing actions of GIP in ob/ob mice. Daily injection of (Pro(3))GIPLys(16)PAL (25 nmol/kg bw) in 14-18-week-old ob/ob mice over 14 days had no effect on body weight, food intake or non-fasting plasma glucose and insulin concentrations. (Pro(3))GIPLys(16)PAL treatment also failed to significantly alter the glycaemic response to an i.p. glucose load or test meal, but insulin concentrations were significantly reduced (1.5-fold; p <0.05) after the glucose load. Insulin sensitivity was enhanced (1.3-fold; p <0.05) and pancreatic insulin was significantly reduced (p <0.05) in the (Pro(3))GIPLys(16)PAL-treated mice. These data demonstrate that acylation of Lys(16) with palmitic acid in (Pro(3))GIP does not improve its biological effectiveness as a GIP receptor antagonist.

Effects of sub-chronic exposure to naturally occurring N-terminally truncated metabolites of glucose-dependent insulinotrophic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), GIP(3-42) and GLP-1 (9-36)amide, on insulin secretion and glucose homeostasis in ob/ob mice

Glucose-dependent insulinotrophic polypepticle (GIP) and glucagon-like peptide-1 (GLP-1) are important enteroendocrine hormones that are rapidly degraded by an ubiquitous enzyme dipeptidyl peptidase IV to yield truncated metabolites GIP(3-42) and GLP-1 (9-36)amide. In this study, we investigated the effects of sub-chronic exposure to these major circulating forms of GIP and GLP-1 on blood glucose control and endocrine pancreatic function in obese diabetic (ob/ob) mice. A once daily injection of either peptide for 14 days had no effect on body weight, food intake or pancreatic insulin content or islet morphology. GLP-1(9-36)amide also had no effect on plasma glucose homeostasis or insulin secretion. Mice receiving GIP(3-42) exhibited small but significant improvements in non-fasting plasma glucose, glucose tolerance and glycaemic response to feeding. Accordingly, plasma insulin responses were unchanged suggesting that the observed enhancement of insulin sensitivity was responsible for the improvement in glycaemic control. These data indicate that sub-chronic exposure to GIP and GLP-1 metabolites does not result in physiological impairment of insulin secretion or blood glucose control. GIP(3-42) might exert an overall beneficial effect by improving insulin sensitivity through extrapancreatic action.

Degradation, cyclic adenosine monophosphate production, insulin secretion, and glycemic effects of two novel N-terminal Ala(2)-substituted analogs of glucose-dependent insulinotropic polypeptide with preserved biological activity in vivo

Glucose-dependent insulinotropic polypeptide (GIP) has significant potential in diabetes therapy due to its ability to serve as a glucose-dependent activator of insulin secretion. However, its biological activity is severely compromised by the ubiquitous enzyme dipeptidylpeptidase IV (DPP IV), which removes the N-terminal Tyr(1)-Ala(2) dipeptide from GIP. Therefore, 2 novel N-terminal Ala(2)-substituted analogs of GIP, with Ala substituted by 2-aminobutyric acid (Abu) or sarcosine (Sar), were synthesized and tested for metabolic stability and biological activity both in vitro and in vivo. Incubation with DPP IV gave half-lives for degradation of native GIP, (Abu(2))GIP, and (Sar(2))GIP to be 2.3, 1.9, and 1.6 hours, respectively, while in human plasma, the half-lives were 6.2, 7.6, and 5.4 hours, respectively. In Chinese hamster lung (CHL) cells expressing the cloned human GIP receptor, native GIP, (Abu(2))GIP, and (Sar(2))GIP dose-dependently stimulated cyclic adenosine monophosphate (camp) production with EC50 values of 18.2, 38.5, and 54.6 nmol/L, respectively. In BRIN-BD11 cells, both (Abu(2))GIP and (Sar(2))GIP (10(-13) to 10(-8) mol/L) dose-dependently stimulated insulin secretion with significantly enhanced effects at 16.7 mmol/L compared with 5.6 mmol/L glucose. In obese diabetic (ob/ob) mice, GIP and (Sar(2))GIP significantly increased (1.4-fold to 1.5-fold; P <.05) plasma insulin concentrations, whereas (Abu(2))GIP exerted only minor effects. Changes in plasma glucose were small reflecting the severe insulin resistance of this mutant. The present data show that substitution of the penultimate N-terminal Ala(2) in GIP by Abu or Sar results in analogs with moderately reduced metabolic stability and biological activity in vitro, but with preserved biological activity in vivo. (C) 2003 Elsevier Inc. All rights reserved.

The combined effect of diabetes and ionising radiation on the retinal vasculature of the rat

The clinical impression that pre-existing diabetes exacerbates radiation injury to the retinal vasculature was studied in STZ diabetic rats. Half of 2 groups of streptozotocin (STZ)-induced diabetic rats and 1 group of normal animals had their right eyes irradiated with 1000 cGy of 90 KVP x-rays. The prevalence of acellular capillaries in trypsin digests of the retinal vasculature was quantified for each of the 6 groups of animals at 6.5 months post-irradiation. The prevalence of acellular capillaries in both non-irradiated diabetic groups was significantly higher than in controls while the irradiated animals in each of the three main categories showed a statistically significant increase compared to their non-irradiated equivalents. However, the net increase in acellular capillaries following irradiation was much greater in rats with an 8 month term of pre-existing diabetes (180%) than in those which had only been diabetic for 3 months (36%). The results of this study suggest a synergistic relationship between pre-existing diabetes and ionising radiation in the development of retinal vasculopathy, and that the potentiation of the vascular damage is dependent on the duration of diabetes prior to radiation exposure.

Regulatory interactions between the metabolic sensors SIRT1 and AMPK in modulating PPARα linked functions, in obesity-dependent type 2 diabetes mellitus

Tese de doutoramento, Ciências Biomédicas (Ciências Funcionais), Universidade de Lisboa, Faculdade de Medicina, 2014

Function of the immunoregulatory CD4-CD8- T cells in the context of autoimmune diabetes

La tolérance immunitaire dépend de la distinction entre le soi et le non soi par le système immunitaire. Un bris dans la tolérance immunitaire mène à l'auto-immunité, qui peut provoquer la destruction des organes, des glandes, des articulations ou du système nerveux central. Le diabète auto-immun, également connu sous le nom diabète juvénile et diabète de type 1, résulte d'une attaque auto-immune sur les cellules β pancréatiques sécrétrices d’insuline, localisées au niveau des îlots de Langerhans du pancréas. Bien que le diabète auto-immun soit traitable par une combinaison d’injections quotidiennes d’insuline d’origine exogène, de régime et d'exercices, beaucoup de complications chroniques peuvent se manifester chez les patients, y compris, mais non limitées à, la cécité, les maladies cardiovasculaires, l’insuffisance rénale et l'amputation. En raison des nombreuses complications liées au diabète auto-immun à long terme, la recherche continue afin de mieux comprendre tous les facteurs impliqués dans la progression de la maladie dans le but de développer de nouvelles thérapies qui empêcheront, renverseront et/ou traiteront cette maladie. Un rôle primordial dans la génération et l'entretien de la tolérance immunitaire a été attribué au nombre et à la fonction des sous-populations de cellules régulatrices. Une de ces populations est constituée de cellules T CD4-CD8- (double négatives, DN), qui ont été étudiées chez la souris et l'humain pour leur contribution à la tolérance périphérique, à la prévention des maladies et pour leur potentiel associé à la thérapie cellulaire. En effet, les cellules de T DN sont d'intérêt thérapeutique parce qu'elles montrent un potentiel immunorégulateur antigène-spécifique dans divers cadres expérimentaux, y compris la prévention du diabète auto-immun. D’ailleurs, en utilisant un système transgénique, nous avons démontré que les souris prédisposées au diabète auto-immun présentent peu de cellules T DN, et que ce phénotype contribue à la susceptibilité au diabète auto-immun. En outre, un transfert des cellules T DN est suffisant pour empêcher la progression vers le diabète chez les souris prédisposées au diabète auto-immun. Ces résultats suggèrent que les cellules T DN puissent présenter un intérêt thérapeutique pour les patients diabétiques. Cependant, nous devons d'abord valider ces résultats en utilisant un modèle non-transgénique, qui est plus physiologiquement comparable à l'humain. L'objectif principal de cette thèse est de définir la fonction immunorégulatrice des cellules T DN, ainsi que le potentiel thérapeutique de celles-ci dans la prévention du diabète auto-immun chez un modèle non-transgénique. Dans cette thèse, on démontre que les souris résistantes au diabète auto-immun présentent une proportion et nombre absolu plus élevés de cellules T DN non-transgéniques, lorsque comparées aux souris susceptibles. Cela confirme une association entre le faible nombre de cellules T DN et la susceptibilité à la maladie. On observe que les cellules T DN éliminent les cellules B activées in vitro par une voie dépendante de la voie perforine et granzyme, où la fonction des cellules T DN est équivalente entre les souris résistantes et prédisposées au diabète auto-immun. Ces résultats confirment que l'association au diabète auto-immun est due à une insuffisance en terme du nombre de cellules T DN, plutôt qu’à une déficience fonctionnelle. On démontre que les cellules T DN non-transgéniques éliminent des cellules B chargées avec des antigènes d'îlots, mais pas des cellules B chargées avec un antigène non reconnu, in vitro. Par ailleurs, on établit que le transfert des cellules T DN activées peut empêcher le développement du diabète auto-immun dans un modèle de souris non-transgénique. De plus, nous observons que les cellules T DN migrent aux îlots pancréatiques, et subissent une activation et une prolifération préférentielles au niveau des ganglions pancréatiques. D'ailleurs, le transfert des cellules T DN entraîne une diminution d'auto-anticorps spécifiques de l'insuline et de cellules B de centres germinatifs directement dans les îlots, ce qui corrèle avec les résultats décrits ci-dessus. Les résultats présentés dans cette thèse permettent de démontrer la fonction des cellules T DN in vitro et in vivo, ainsi que leur potentiel lié à la thérapie cellulaire pour le diabète auto-immun.

The component polypeptide chains of bovine insulin nucleate or inhibit aggregation of the parent protein in a conformation-dependent manner

Amyloid fibrils are typically rigid, unbrariched structures with diameters of similar to 10 nm and lengths up to several micrometres, and are associated with more than 20 diseases including Alzheimer's disease and type II diabetes. Insulin is a small, predominantly alpha-helical protein consisting of 51 residues in two disulfide-linked polypeptide chains that readily assembles into amyloid fibrils under conditions of low PH and elevated temperature. We demonstrate here that both the A-chain and the B-chain of insulin are capable of forming amyloid fibrils in isolation under similar conditions, with fibrillar morphologies that differ from those composed of intact insulin. Both the A-chain and B-chain fibrils were found to be able to cross-seed the fibrillization of the parent protein, although these reactions were substantially less efficient than self-seeding with fibrils composed of full-length insulin. In both cases, the cross-seeded fibrils were morphologically distinct from the seeding, material, but shared common characteristics with typical insulin fibrils, including a very similar helical repeat. The broader distribution of heights of the cross-seeded fibrils compared to typical insulin fibrils, however, indicates that their underling protofilament hierarchy may be subtly different. In addition, and remarkably in view of this seeding behavior, the soluble forms of the A-chain and B-chain peptides were found to be capable of inhibiting insulin fibril formation. Studies using mass spectrometry suggest that this behavior might be attributable to complex formation between insulin and the A-chain and B-chain peptides. The finding that the same chemical form of a polypeptide chain in different physical states can either stimulate or inhibit the conversion of a protein into amyloid fibrils sheds new light on the mechanisms underlying fibril formation, fibril strain propagation and amyloid disease initiation and progression. (c) 2006 Elsevier Ltd. All rights reserved.

The effects of lipoic acid and alpha-tocopherol supplementation on the lipid profile and insulin sensitivity of patients with type 2 diabetes mellitus: A randomized, double-blind, placebo-controlled trial

Antioxidants probably play an important role in the etiology of type 2 diabetes (DM2). This study evaluated the effects of supplementation with lipoic acid (LA) and alpha-tocopherol on the lipid profile and insulin sensitivity of DM2 patients. A randomized, double-blind, placebo-controlled trial involving 102 DM2 patients divided into four groups to receive daily supplementation for 4 months with: 600 mg LA (n = 26); 800 mg alpha-tocopherol (n = 25); 800 mg alpha-tocopherol + 600 mg LA (n = 25); placebo (n = 26). Plasma alpha-tocopherol, lipid profile, glucose, insulin, and the HOMA index were determined before and after supplementation. Differences within and between groups were compared by ANOVA using Bonferroni correction. Student`s t-test was used to compare means of two independent variables. The vitamin E/total cholesterol ratio improved significantly in patients supplemented with vitamin E + LA and vitamin E alone (p <= 0.001). There were improvements of the lipid fractions in the groups receiving LA and vitamin E alone or in combination, and on the HOMA index in the LA group, but not significant. The results suggest that LA and vitamin E supplementation alone or in combination did not affect the lipid profile or insulin sensitivity of DM2 patients. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

Microalbuminuria is associated with impaired arterial and venous endothelium-dependent vasodilation in patients with Type 2 diabetes

Background: Microalbuminuria in Type 2 diabetes is associated with arterial endothelial dysfunction, but the venous bed was never evaluated. Aim: To study the endothelial function in the venous and arterial bed in patients with Type 2 diabetes with normoalbuminuria or microalbuminuria. Material and methods: We evaluated 28 patients with Type 2 diabetes, glycated hemoglobin (Hbak(1c)) <7.5%, who were classified as normo- (albuminuria <30 mg/24 h; no.=16) or microalbuminuric (albuminuria 30-300 mg/24 h; no.=12). Venous and arterial endothelial function were assessed by the dorsal hand vein technique (venodilation by acetylcholine) and brachial artery flow-mediated vasodilation, respectively. Results: Patients were normotensive (systolic arterial pressure: 131.1 +/- 10.6 mmHg) and on good metabolic control (HbA(1c): 6.6 +/- 0.6%). Microalbuminuric patients presented impaired venous (32.9 +/- 17.4 vs 59.3 +/- 26.5%; p=0.004) and arterial vasodilation (1.8 +/- 0.9 vs 5.1 +/- 2.4; p<0.001), as compared to normoalbuminuric patients. There was a negative correlation between acetylcholine-induced venodilation and albuminuria (r=-0.62; p<0.001) and HbA(1c) (r=-0.41; p=0.032). The same was observed between flow-mediated arterial vasodilation and albuminuria (r=-0.49; p=0.007) and HbA(1c) (r=-0.44; p=0.019). Venous and arterial vasodilation was positively correlated (r=0.50; p=0.007). Conclusions: Both venous and arterial endothelial function are impaired in Type 2 microalbuminuric diabetics, in spite of good metabolic control, suggesting that other factors are involved in its pathogenesis. (J. Endocrinol. Invest. 33: 696-700, 2010) (C) 2010, Editrice Kurtis