Angiotensin blockade and matrix synthesis by glomerular epithelial cells in high glucose: a further experimental insight into the pathophysiology of diabetic nephropathy.

AIMS: Angiotensin converting enzyme inhibitors (ACEIs) and angiotensin II receptor-1 (AT-1) antagonists are used in the treatment of proteinuria of diabetic nephropathy. One of the major pathogenic events in this condition is represented by the alteration of the extracellular matrix protein synthesis by glomerular epithelial cells. MATERIALS AND METHODS: We evaluated the effects of the angiotensin converting enzyme inhibitor, Enalaprilat, and the AT-1 receptor antagonist, Losartan, on mRNA fibronectin and laminin synthesis by glomerular epithelial cells, in conditions mimicking hyperglycemia. RESULTS: In high glucose conditions, Enalaprilat reduced significantly the mRNA expression of fibronectin (p 0.03), but not significantly that of laminin. Losartan addition to high glucose incubated cells reduced (-30%) mRNA expression of fibronectin, and significantly (p 0.05) the mRNA expression of laminin. CONCLUSIONS: In addition to the known hemodynamic effects, the improvement of renal function in diabetic patients treated with these compounds may also be due to a modulator effect on extracellular matrix content and composition.

Oral probiotic administration induces interleukin-10 production and prevents spontaneous autoimmune diabetes in the non-obese diabetic mouse.

AIMS/HYPOTHESIS: Recent observations suggest the involvement of the gastrointestinal tract in the pathogenesis of islet autoimmunity. Thus, the modulation of gut-associated lymphoid tissue may represent a means to affect the natural history of the disease. Oral administration of probiotic bacteria can modulate local and systemic immune responses; consequently, we investigated the effects of oral administration of the probiotic compound VSL#3 on the occurrence of diabetes in non-obese diabetic (NOD) mice. METHODS: VSL#3 was administered to female NOD mice three times a week starting from 4 weeks of age. A control group received PBS. Whole blood glucose was measured twice a week. IFN-gamma and IL-10 production/expression was evaluated by ELISA in culture supernatants of mononuclear cells isolated from Peyer's patches and the spleen, and by real-time PCR in the pancreas. Insulitis was characterised by immunohistochemistry and histomorphometric studies. RESULTS: Early oral administration of VSL#3 prevented diabetes development in NOD mice. Protected mice showed reduced insulitis and a decreased rate of beta cell destruction. Prevention was associated with an increased production of IL-10 from Peyer's patches and the spleen and with increased IL-10 expression in the pancreas, where IL-10-positive islet-infiltrating mononuclear cells were detected. The protective effect of VSL#3 was transferable to irradiated mice receiving diabetogenic cells and splenocytes from VSL#3-treated mice. CONCLUSIONS/INTERPRETATION: Orally administered VSL#3 prevents autoimmune diabetes and induces immunomodulation by a reduction in insulitis severity. Our results provide a sound rationale for future clinical trials of the primary prevention of type 1 diabetes by oral VSL#3 administration.

The acquisition of an insulin-secreting phenotype by HGF-treated rat pancreatic ductal cells (ARIP) is associated with the development of susceptibility to cytokine-induced apoptosis.

The elucidation of mechanisms regulating the regeneration and survival of pancreatic beta cells has fundamental implications in the cell therapy of type 1 diabetes. The present study had the following three aims: 1. to investigate whether pancreatic ductal epithelial cells can be induced to differentiate into insulin-producing cells by exposing them to hepatocyte growth factor (HGF); 2. to characterize some of the molecular events leading to their differentiation toward a beta-cell-like phenotype; 3. to evaluate the susceptibility of newly differentiated insulin-secreting cells to cytokine-induced apoptosis, a mechanism of beta-cell destruction occurring in type 1 diabetes. We demonstrated that HGF-treated rat pancreatic ductal cell line (ARIP) cells acquired the capability to transcribe the insulin gene and translate its counterpart protein. HGF-treated cells also exhibited a glucose-dependent capability to secrete insulin into the cultured medium. Expression analysis of some of the genes regulating pancreatic beta-cell differentiation revealed a time-dependent transcription of neurogenin-3 and Neuro-D in response to HGF. Finally, we determined the susceptibility to proinflammatory cytokine (PTh1)-induced apoptosis by incubating HGF-treated and untreated ARIP cells with a cocktail of interleukin-1 beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). Such treatment induced apoptotic death, as determined by the TUNEL technique, in about 40% of HGF-treated, insulin-secreting ARIP cells, while untreated ARIP cells were resistant to PTh1-induced apoptosis. In conclusion, we showed that HGF promotes the differentiation of ARIP cells into pancreatic beta-cell-like cells, and that the differentiation toward an insulin-secreting phenotype is associated with the appearance of susceptibility to cytokine-induced apoptosis.

Acute hyperinsulinemia is associated with increased plasma adrenomedullin concentrations in uncomplicated obesity.

OBJECTIVE: Adrenomedullin (AM) is a potent hypotensive peptide which may be implicated in the insulin regulatory system. Acute hyperinsulinemia exerts no influence on plasma AM in normal subjects while no data on obese subjects has been reported. PURPOSE: The aim of the study was to investigate the effect of acute hyperinsulinemia on the plasma AM concentration in patients with uncomplicated obesity. RESEARCH METHODS: We measured the plasma AM levels in 23 obese subjects (BMI 41.9 +/- 9.8 kg/m2), 21 females and 2 males (mean age 31 +/- 7.2 years), before and during a euglycemic hyperinsulinemic clamp. The control group consisted of 43 healthy subjects (HS) (22 males and 21 females; mean age 38 +/- 12 years; BMI 23.3 +/- 3.2 kg/m2). RESULTS: Baseline plasma AM was found to be higher in obese subjects (20.4 +/- 8.4 pg/ml) than in normal subjects (11.3 +/- 0.8 pg/ml) (p < 0.001). A significant increase in the plasma AM levels was observed in obese subjects during acute hyperinsulinemia (from 20.4 +/- 8.4 pg/ml at 0 min to 26 +/- 8.9 pg/ml at 120 min, p < 0.02). Plasma AM concentrations were significantly correlated with insulin levels at 30 min (r = 0.44; p = 0.04) and 120 min (r = 0.40, p = 0.05) during the clamp. DISCUSSION: In conclusion, acute hyperinsulinemia induced a significant increase in the plasma levels of AM in uncomplicated obese subjects. Hyperinsulinemia may, at least in part, regulate levels of AM in obesity, explaining the high levels of the peptide in these subjects.

GUIDE study: double-blind comparison of once-daily gliclazide MR and glimepiride in type 2 diabetic patients.

BACKGROUND: Progressive beta-cell failure is a characteristic feature of type 2 diabetes; consequently, beta-cell secretagogues are useful for achieving sufficient glycaemic control. The European GUIDE study is the first large-scale head-to-head comparison of two sulphonylureas designed for once-daily administration used under conditions of everyday clinical practice. DESIGN: Eight hundred and forty-five type 2 diabetic patients were randomized to either gliclazide modified release (MR) 30-120 mg daily or glimepiride 1-6 mg daily as monotherapy or in combination with their current treatment (metformin or an alpha-glucosidase inhibitor) according to a double-blind, 27-week, parallel-group design. Efficacy was evaluated by HbA1c and safety by hypoglycaemic episodes using the European Agency definition. RESULTS: HbA1c decreased similarly in both groups from 8.4% to 7.2% on gliclazide MR and from 8.2% to 7.2% on glimepiride. Approximately 50% of the patients achieved HbA1c levels less than 7%, and 25% less than 6.5%. The mean difference between groups of the final HbA1c was -0.06% (noninferiority test P < 0.0001). No hypoglycaemia requiring external assistance occurred. Hypoglycaemia with blood glucose level < 3 mmol L(-1) occurred significantly less frequently (P = 0.003) with gliclazide MR (3.7% of patients) compared with glimepiride (8.9% of patients). The distribution of the sulphonylurea doses was similar in both groups. CONCLUSIONS: This study provides new insights into therapeutic strategies using sulphonylureas. It shows that gliclazide MR is at least as effective as glimepiride, either as monotherapy or in combination. The safety of gliclazide MR was significantly better, demonstrating approximately 50% fewer confirmed hypoglycaemic episodes in comparison with glimepiride.

The impairment of renal function is not associated to altered circulating vascular endothelial growth factor in patients with Type 2 diabetes and hypertension.

BACKGROUND: Metabolic and haemodynamic factors concur in the development of diabetic nephropathy. Moreover, in diabetes, the presence of hypertension may accelerate the development of renal damage. Vascular endothelial growth factor (VEGF) stimulates microvascular permeability, endothelium-dependent vasodilation and angiogenesis and its synthesis is enhanced by hyperglycaemia, advanced glycation end-products (AGEs), tissue hypoxia and hypertension. VEGF appears to play a central role in mediating diabetic vasculopathy, and although VEGF and its receptors are expressed at renal level, its action in renal pathophysiology is unknown. The aim of this study was to elucidate whether presence and/or severity of renal dysfunction is related to circulating VEGF in patients with Type 2 diabetes and hypertension. DESIGN AND METHODS: Fifty hypertensive Type 2 diabetic patients and 20 non-diabetic patients were included in the study. Renal function parameters such as albumin excretion rate (AER), and glomerular filtration rate (GFR), and VEGF plasma levels were analysed in all subjects, whereas %HbA1c and AGEs levels were evaluated in diabetic patients. RESULTS: GFR was significantly decreased in diabetic patients compared with the control subjects (74.36 +/- 15.95 vs 111.5 +/- 17.0 ml/min, p<0.0001). Three diabetic patients showed AER abnormalities (53.8 +/- 2.3 mg/24h). VEGF in diabetic patients was higher than in the control group (77.95 +/- 65.98 vs 49.30 +/- 40.8 pg/ml), but not significantly. %HbA1c and AGE levels were 6.6 +/- 1.5% and 11.59 +/- 8.09 UAGE/ml, respectively. No correlation was found between renal function, circulating VEGF levels and metabolic control. CONCLUSION: Diabetes, in association with hypertension, significantly decreases renal function, but circulating VEGF may not reflect its concentration and action at renal level.

Genetic prediction of type 1 diabetes in a population with low frequency of HLA risk genotypes and low incidence of the disease (the DIABFIN study).

BACKGROUND: To develop a sensitive, specific screening strategy for predicting genetic risk for type 1 diabetes mellitus (T1DM) in the low-incidence continental Italian population, and to define with this tool, a cohort of high-to-moderate risk infants for an immunological follow-up study aimed at identifying environmental risk factors for T1DM. METHODS: 4855 newborns in three regions of continental Italy were screened for T1DM HLA-DRB1-DQB1 risk genotypes using a reverse line blot typing method. Risk classification was based on odds ratios (OR) found in a preliminary case-control study (356 T1DM patients, 412 controls). Screening efficiency was optimized by allele subtyping. RESULTS: Screening for well-known T1DM susceptibility genotypes [DRB1*03/*04-DQB1*0302; DRB1*03/*03; DRB1*04/*04-DQB1*0302; DRB1*04-DQB1*0302/X where X is not equal to DRB1*03, DRB1*04-DQB1*0302, DQB1*0602 or DQB1*0603] was associated with <60% sensitivity due to their low frequencies in the general Italian population. Inclusion of an additional genotype from which protective DRB1 and DQB1 alleles had been excluded [DRB1*03/X degrees where DQB1 is not equal to *0301, *0503, *0602, or *0603 and X degrees not equal DRB1*03, DRB1*04-DQB1*0302 or DRB1*07] increased screening sensitivity to 75% (specificity: 85%). Among 4855 newborns, we have found the high-risk genotype [DRB1*03/*04-DQB1*0302; estimated absolute risk (AR) 1/23] to be present in only 0.9%. The moderate-risk genotypes were found in 13.8% of newborns (estimated AR 1/177). CONCLUSIONS: Risk classification must be tailored to the characteristics of the individual population, in particular, the allelic frequencies in the background population and T1DM prevalence. We have developed a screening strategy with good levels of sensitivity that should prove effective for use throughout the Italian peninsula.

Insulin sensitivity assessment in uncomplicated obese women: comparison of indices from fasting and oral glucose load with euglycemic hyperinsulinemic clamp.

BACKGROUND AND AIM: Obesity is associated with a great variability to insulin sensitivity degree. Several formulae developed from measurements in the fasting state and during the oral glucose tolerance test (OGTT) have been proposed to assess insulin sensitivity. AIM: In this work we sought to compare the published insulin sensitivity indices with the metabolized glucose index obtained by hyperinsulinemic euglycemic clamp in uncomplicated obese subjects. Uncomplicated obesity provides a good model in order to study insulin sensitivity per se. METHODS AND RESULTS: In this protocol, 65 obese women affected by uncomplicated obesity (without impaired glucose tolerance, diabetes, hypertension and dyslipidemia) underwent 2 h OGTT and euglycemic hyperinsulinemic clamp. Common formulae obtained in the fasting state and from a 2h OGTT were calculated. Simple linear regression analysis showed that ISI (r=0.592, p=0.01), 2 h OGIS (r=0.576, p=0.02), MCRest (r=0.507, p=0.02), 120 insulin (r=-0.494, p=0.03) and fasting insulin (r=-0.382, p =0.02) are significantly correlated to the M index obtained from the hyperinsulinemic euglycemic clamp. The Bland-Altman plot confirmed the good agreement between indices from OGTT and the clamp. CONCLUSION: OGTT-derived indices provide a good assessment of insulin sensitivity in obesity. OGTT could easily be applied in a large number of obese patients in order to obtain information on both glucose tolerance and insulin sensitivity.

The G972R variant of the insulin receptor substrate-1 (IRS-1) gene is associated with insulin resistance in "uncomplicated" obese subjects evaluated by hyperinsulinemic-euglycemic clamp.

Several association studies have indicated the insulin receptor substrate-1 (IRS-1) gene G972R variant as a genetic risk factor for insulin resistance, particularly in presence of obesity. A few studies have also suggested a possible effect of the G972R variant on insulin secretion. The aim of this study was to evaluate the role of the IRS-1 gene G972R variant in 61 subjects with "uncomplicated" obesity [i.e. without diabetes, hypertension, dyslipidemia, coronary artery disease (CAD)], studied by hyperinsulinemic-euglycemic clamp. The presence of the G972R variant, detected in real-time with LightCycler hybridisation probes, was related to the indexes of insulin sensitivity. Furthermore, the possible role of this variant on insulin secretion was studied by means of insulin release indexes derived from oral tolerance test (OGTT). Twenty-four point five percent (24.5%) (no.=15) of the obese subjects proved to be carriers of the G972R variant. M index (p<0.05), non-oxidative glucose (p<0.01), insulin clearance (p<0.03) and insulin sensitivity index (ISI) (p<0.005) were all significantly reduced in G972R carriers compared to non-carriers, indicating a significant reduction in insulin sensitivity in carriers of the variant. A logistic regression analysis confirmed the independent association between the G972R variant and reduced insulin sensitivity (p<0.03). The interaction between obesity and the G972R variant was also independently associated with a reduced insulin sensitivity (p<0.005), suggesting that obesity and G972R variant were more than additive in predicting insulin resistance. The analysis of insulin release indexes did not show any significant differences. Our results demonstrate the association of the G972R variant of the IRS-1 gene with reduced insulin sensitivity in obese subjects, and indicate a possible interaction between the IRS-1 variant and obesity in worsening of insulin sensitivity.

Celiac disease-associated transglutaminase autoantibody target domains at diagnosis are age and sex dependent.

The contribution of age and/or sex to the transglutaminase (tTG) autoantibody response in celiac disease (CD) is not known. To gain insights into transglutaminase humoral autoimmunity at CD diagnosis, our aim was to characterize the autoimmune response against three tTG constructs [(full-length tTG(a.a.1-687), tTG(a.a.227-687), and tTG(a.a.473-687)] and to investigate into its relationship with CD patients' age and sex. One hundred seventy-five newly diagnosed CD patients (115 females and 60 males), subdivided into different groups according to age and sex, were studied using a serum 35S-radioimmunoassay. We found that among full-length tTG autoantibody-positive CD subjects (175/175), 50.9% (89/175) and 83.4% (146/175) had autoantibodies against tTG(227-687) and tTG(473-687) domains, respectively. Female patients of less than 4 years expressed tTG(227-687)Abs in significantly higher percentage and mean autoantibody titers vs. all other groups investigated, and tTG(473-687)Abs in significantly higher titers with respect to adult female patients. Our data identify a subset of CD patients showing a strong humoral tTG immunoreactivity at diagnosis, thus suggesting that age and sex influence the anti-tTG autoantibody response.

Angiotensin-converting enzyme inhibition modulates high-glucose-induced extracellular matrix changes in mouse glomerular epithelial cells.

BACKGROUND/AIM: Extracellular matrix alterations are involved in the pathogenesis of diabetic nephropathy. We evaluated the effects of high glucose concentrations and inhibition of angiotensin-converting enzyme on the laminin and fibronectin production by glomerular epithelial cells. METHODS: Glomerular epithelial cells were cultured in 5 and 30 mmol/l glucose, with and without enalaprilat (0.3 mmol/l). Laminin and fibronectin were measured (35S-methionine, immunoprecipitation), and their mRNA expression was evaluated (RT-PCR). RESULTS: The laminin concentration was higher in the cells than in the medium, where an increase of its content was observed under high-glucose conditions (p < 0.01). Fibronectin, found only in the medium, was not modified by the high glucose concentration. Following enalaprilat administration, the laminin concentration was decreased under high-glucose conditions, both in the cell and in the medium (p < 0.001), whereas the fibronectin concentration was increased under high-glucose conditions (p < 0.001). The mRNA expression of laminin and fibronectin under high-glucose conditions only slightly increased. Enalaprilat decreased the fibronectin mRNA synthesis dramatically (>50%, p < 0.0001) under high-glucose conditions. CONCLUSIONS: Enalaprilat normalizes the abnormal, high-glucose-induced concentration of laminin, while it decreases the fibronectin synthesis. The improvement of the renal function in diabetic patients treated with angiotensin-converting enzyme inhibitors may, in part, be due to a modulator effect on extracellular matrix content and composition.

Rats that are made insulin resistant by glucosamine treatment have impaired skeletal muscle insulin receptor phosphorylation.

The current study sought to verify whether glucosamine (GlcN)-induced insulin resistance is associated with impaired insulin receptor (IR) autophosphorylation. Rats were given either saline or primed continuous GlcN infusion (5 micromol x kg(-1) x min(-1)) 10 minutes prior to and during euglycemic hyperinsulinemic clamp (primed continuous infusion of 20 mU x kg(-1) x min(-1) insulin for 2 hours). IR autophosphorylation was measured in skeletal muscle after in vivo insulin stimulation (ie, during clamp) by Western blot and then retested after subsequent in vitro 0.1 to 100 nmol/L insulin stimulation (by enzyme-linked immunosorbent assay [ELISA]). Tissue PC-1 enzymatic activity was also measured. In vivo, insulin/GlcN rats had decreased (P <.01) whole body glucose uptake (37.7 +/- 2.1 v 49.7 +/- 2.7 mg x kg(-1) x min(-1) in respect to insulin/saline), receptor autophosphorylation (37 +/- 5 v 82 +/-.0 arbitrary units/mg protein), and insulin receptor substrate-1 (IRS-1) phosphorylation (112% +/- 15% v 198% +/- 23% of saline infusion rats). Receptor autophosphorylation was correlated with whole body glucose uptake (r = 0.62, P <.05). Skeletal muscle PC-1 activity (58.8 +/- 10.7 v 55.7 +/- 5.8 nmol x mg(-1) x min(-1)) was not different in the 2 groups. Our data show that GlcN-induced insulin resistance is mediated, at least in part, by impaired skeletal muscle IR autophosphorylation.

Type 1 diabetes islet associated antibodies in subjects infected by echovirus 16.

AIMS/HYPOTHESIS: To determine whether the emergent infection by echovirus 16 that occurred in Cuba during the year 2000 was related to the presence of Type 1 diabetes associated autoantibodies. METHODS: The presence of ICA, IAA, GADA, IA2 antibodies and neutralizing antibodies (NtAb) to echovirus 16 were determined in sera from 38 infected children and adolescents and 80 control subjects, matched in sex, age, local residence and time of sample collection. RESULTS: The occurrence of a large-scale echovirus 16 epidemic was associated with the appearance of humoral autoimmune markers of Type 1 diabetes, especially for ICA, IAA and GADA. In the convalescent stage, ICA, IAA and GADA seroconversion was shown in 92.1%, 44.7% and 28.9% of echovirus 16 infected subjects. None of the 80 uninfected subjects had ICA or IAA, while one was GADA positive. ICA, IAA and GADA frequency was higher in the convalescent than in the acute stage (p<0.0005). A strong positive correlation was found between the NtAb to echovirus 16 and ICA titres in both acute and convalescent stage (r=0.91; p<0.0001, r=0.55; p=0.0003 respectively). CONCLUSION/INTERPRETATION: This work provides evidence of an association between echovirus 16 infection and the presence of Type 1 diabetes related antibodies (ICA, IAA and GADA). Our data show that the echovirus 16 infection might be capable of inducing a process of autoimmune beta-cell damage and support the hypothesis that enterovirus infections are important risk factors for the development of Type 1 diabetes.

Two polyclonal antisera detect different AGE epitopes in human plasma samples.

Advanced glycation end products (AGEs), involved in the pathogenesis of diabetic complications, comprise a series of related chemical structures which might possess dissimilar immunogenic characteristics. In this study the levels of AGE in plasma samples from normal subjects (N=41) and diabetic patients (N=44) were measured by ELISA using two polyclonal antisera (named CF5 and CF199, respectively, and immunologically characterized) raised using two different immunogens and immunization techniques. Age levels were significantly higher in diabetic than in normal plasma samples (P<0.0001) with both antisera. However, CF199 detected higher AGE levels than CF5 both in normal (P<0.0001) and diabetic (P<0.005) samples. Pre-incubation with AGE-bovine serum albumin (BSA) caused the loss of most the reactivity of both antisera. Pre-incubation with carboxy-methyl-lysine-BSA (an oxidation-derived AGE) induced the loss of nearly all CF5 reactivity while CF199 retained a significant amount of activity against AGE antigens. Moreover, CF5 lost over 90% of its reactivity against BSA incubated with high glucose under non-oxidative conditions, suggesting its recognition of mainly oxidation-derived AGE epitopes. The different AGE levels measured by the two antisera suggests, therefore, that one single antiserum is unable to recognize all the various AGE epitopes which might be present, at any time, in tissues and body fluids in health and disease.

Importance of premeal injection time in insulin therapy: Humalog Mix25 is convenient for improved post-prandial glycemic control in type 2 diabetic patients with Italian dietary habits.

We investigated the use, in a short period, of Humalog Mix25 (Mix25) in a twice-daily administration regimen compared to a twice-daily injection therapy with Humulin 30/70 (30/70) in diabetic patients with Italian dietary habits. We studied 33 type 2 diabetic patients aged 59.1 +/- 8.1 years, BMI 29.8 +/- 2.7 kg/m2, duration of diabetes and insulin therapy of 14.4 +/- 9.8 and 4.2 +/- 4.6 years, respectively. After a 4-day lead-in period of twice-daily human insulin 30/70 treatment, patients were randomized to one of two treatment sequences: (1) a twice-daily regimen with Mix25 just 5 minutes before the morning and evening meals for 12 days, followed by a twice-daily therapy with human insulin 30/70 given 30 minutes before the morning and evening meals for an additional 12 days; or (2) the alternate sequence. Each patient underwent a mixed meal test: Humulin 30/70 was administered 30 minutes before the meal, while Mix25 was given 5 minutes before. The 2-hour post-prandial glucose concentration after breakfast was significantly lower during treatment with Mix25 than with Humulin 30/70 (157 +/- 43.2 vs. 180 +/- 43.2 mg/dl, p<0.05). The glycemic excursion after dinner on Mix25 treatment was significantly lower than with Humulin 30/70 (12.2 +/- 48.01 vs. 35.5 +/- 36.92 mg/dl, p<0.05). AUCglucose after Mix25 was lower than after Humulin 30/70. Glycemia after test meal was significantly lower with Mix25 than with Humulin 30/70. Insulin and free insulin concentrations after the test meal were significantly higher with Mix25 in comparison to Humulin 30/70. AUC serum insulin and free insulin curves after Mix25 were significantly higher than after Humulin 30/70 (p=0.028 and p=0.005, respectively). Twice-daily injections of Humalog Mix25, compared to human insulin 30/70 in type 2 diabetic patients with Italian dietary habits, provide improved and lasting post-prandial glycemic control, with the great convenience of the injection just before the meal.

Cultured pancreatic ductal cells undergo cell cycle re-distribution and beta-cell-like differentiation in response to glucagon-like peptide-1.

The intestinal hormone glucagon-like peptide-1 (GLP-1) has been shown to promote an increase in pancreatic beta-cell mass via proliferation of islet cells and differentiation of non-insulin-secreting cells. In this study, we have characterized some of the events that lead to the differentiation of pancreatic ductal cells in response to treatment with human GLP-1. Rat pancreatic ductal (ARIP) cells were cultured in the presence of GLP-1 and analyzed for cell counting, cell cycle distribution, expression of cyclin-dependent-kinase (Cdk) inhibitors, transcription of beta-cell-specific genes, loss of ductal-like phenotype and acquisition of beta-cell-like gene expression profile. Exposure of ARIP cells to 10 nM GLP-1 induced a significant reduction in the cell replication rate and a significant decrease in the percentage of cells in S phase of the cell cycle. This was associated with an increase in the number of cells in G0-G1 phase and a reduction of cells in G2-M phase. Western blot analysis for the Cdk inhibitors, kinase inhibitor protein 1 (p27(Kip1)) and Cdk-interacting protein 1 (p21(Cip1)), demonstrated a significant increase in p27(Kip1) and p21(Cip1) levels within the first 24 h from the beginning of GLP-1 treatment. As cells slowed down their proliferation rate, GLP-1 also induced a time-dependent expression of various beta-cell-specific mRNAs. The glucose transporter GLUT-2 was the first of those factors to be expressed (24 h treatment), followed by insulin (44 h) and finally by the enzyme glucokinase (56 h). In addition, immunocytochemistry analysis showed that GLP-1 induced a time-dependent down-regulation of the ductal marker cytokeratin-20 (CK-20) and a time-dependent induction of insulin expression. Finally, GLP-1 promoted a glucose-dependent secretion of insulin, as demonstrated by HPLC and RIA analyses of the cell culture medium. The present study has demonstrated that GLP-1 induces a cell cycle re-distribution with a decrease in cell proliferation rate prior to promoting the differentiation of cells towards an endocrine-like phenotype.

Clinical, physiopathological and dietetic aspects of metabolic syndrome.

Hypertriglyceridaemia, diabetes, hypertension and obesity are the deadly quartet indicating a syndrome at high risk for cardiovascular disease for which, in 1998, WHO proposed the definition of Metabolic Syndrome, related to an elevated degree of insulin resistance. Treatment will often include behavioural changes that reduce body weight and increase physical activity A high-carbohydrate/low-fat diet with complex carbohydrates and mainly unsaturated fat is recommended. Replacing refined grain products and potatoes with minimally processed plant-based foods such as whole grains, fruit, and vegetables, and reducing the intake of high glycaemic load beverages may offer a simple strategy for reducing the incidence of coronary heart disease.

Similar incidence of type 1 diabetes in two ethnically different populations (Italy and Slovenia) is sustained by similar HLA susceptible/protective haplotype frequencies.

The incidence of type 1 diabetes (T1DM) seems to depend in part on the population frequencies of susceptible and protective HLA haplotypes. The present study aimed to (i): characterize the genetic susceptibility to T1DM in the Slovenian population, (ii) test the general hypothesis that T1DM incidence is related to the frequencies of susceptible/protective haplotypes, (iii) compare allele, haplotype and genotype frequencies in Slovenians and Italians that represent two white populations with a similar incidence of T1DM (7.9/100,000/year and 8.1/100,000/year, respectively). The haplotype found most frequently among Slovenian T1DM patients was DRB1*0301-DQA1*0501-DQB1*0201 (53%). The DR4-DQA1*0301-DQB1*0302 haplotypes conferring susceptibility to T1DM were those bearing DRB1*0401 (OR = 12), DRB1*0404 (OR = 4.7) and DRB1*0402 (OR = 4.5). Negative associations with the disease were found for the following haplotypes: DRB1*1501-DQA1*0102-DQB1*0602, DRB1*1301-DQA1*0102-DQB1*0603, DRB1*1101/1104-DQA1*0501-DQB1*0301, and DRB1*1401-DQA1*0101-DQB1*0503. Our findings indicate that the low frequencies of susceptible genotypes, in particular, DR3-DQA1*0501-DQB1*0201/DR4-DQA1*0301-DQB1*0302, together with a high frequency of protective haplotypes, could in part explain the low incidence of T1DM in the Slovenian population. The combined frequencies of susceptible genotypes were similar in the two populations (Slovenia = 19.2%, Italy = 17.6%), and the 95% confidence limits of the OR values for each genotype in the two populations overlapped, indicating no significant differences between the values. We conclude that the similar incidences of T1DM in Italian and Slovenian populations are in part a reflection of similar frequencies of HLA susceptible/protective haplotypes.

Metformin decreases platelet superoxide anion production in diabetic patients.

BACKGROUND: Patients with type 2 diabetes mellitus are usually treated with oral antidiabetic agents but it is still not known whether these drugs have antioxidant effects in humans. METHODS: We studied 60 patients with type 2 diabetes mellitus, divided into three groups on the basis of hypoglycaemic treatment (Group A: metformin, Group B: glibenclamide, Group C: diet). All patients were followed for at least 1 year. The three subgroups had similar clinical characteristics. Twenty healthy subjects, of comparable sex and age, were enrolled as controls. In each subject, platelet production of superoxide anion (O(2)(-)) elicited by collagen, was determined by lucigenin assay. RESULTS: Patients with diabetes had higher platelet O(2)(-) production than controls; no correlation was observed between blood glucose and platelet O(2)(-) production. Group A patients had platelet O(2)(-) production similar to that of healthy subjects but lower than Group B and Group C patients. CONCLUSION: The present findings suggest an in vivo antioxidant activity of metformin and warrant prospective studies to further explore this hypothesis.