Pioglitazone attenuates the detrimental effects of advanced glycation end-products in the pancreatic beta cell line HIT-T15.

Pioglitazone is an anti-diabetic agent that preserves pancreatic beta cell mass and improves their function. Advanced Glycation End-Products (AGEs) are implicated in diabetic complications. We previously demonstrated that exposure of the pancreatic islet cell line HIT-T15 to high concentrations of AGEs significantly decreases cell proliferation and insulin secretion, and affects transcription factors regulating insulin gene transcription. The aim of this work was to investigate the effects of Pioglitazone on the function and viability of HIT-T15 cells cultured with AGEs. HIT-T15 cells were cultured for 5 days in the presence of AGEs alone, or supplemented with 1 µmol/l Pioglitazone. Cell viability, insulin secretion and insulin content, redox balance, expression of the AGE receptor (RAGE), and NF-kB activation were then determined. The results showed that Pioglitazone protected beta cells against AGEs-induced apoptosis and necrosis. Moreover, Pioglitazone restored the redox balance and improved the responsiveness to low glucose concentration. Adding Pioglitazone to the AGEs culture attenuated NF-kB phosphorylation, and prevented AGEs to down-regulate IkBα expression. These findings suggest that Pioglitazone protects beta cells from the dangerous effects of AGEs.

Recommendations for the use of cardiovascular tests in diagnosing diabetic autonomic neuropathy.

Despite its prevalence, clinical and prognostic impact, diabetic autonomic neuropathy, is widely under-diagnosed. The need for training and expertise to perform the cardiovascular tests (usually the task of diabetologists) is one possible reason. The availability of computer-assisted systems has allowed a wider diffusion of testing, but has also highlighted the need for an adequate knowledge of physiopathological backgrounds for their correct application and interpretation. The recommendations presented here were developed by the Neuropathy Study Group of the Italian Society of Diabetology and then endorsed by the Italian Association for the Study of Neurovegetative System, to promote the widespread adoption of good clinical practice in diabetic cardiovascular autonomic testing by outlining main evidence-based aspects, i.e. which tests, how to perform them, adequate interpretation of the results and their diagnostic use, confounding conditions that can impact on tests reliability. Therefore, these recommendations include the essential aspects of the physiopathological substrate of the tests, the controversial points in their analysis, their diagnostic characteristics, as well as safety. Detailed information is given on the physiological (age, weight, body position, resting heart rate and blood pressure, respiratory pattern, exercise, meals, acute blood glucose changes) and pathophysiological confounding factors, with emphasis on the effects of drugs. Instructions on how to perform the tests and interpret their results are also considered together with indications of candidate patients and periodicity of testing. A patient instruction sheet on why and how to perform the tests is included. Finally, the specific requirements for computerized systems to perform and evaluate cardiovascular tests are provided.

Glucagon-like peptide-1 counteracts the detrimental effects of Advanced Glycation End-Products in the pancreatic beta cell line HIT-T 15.

Advanced Glycation End-Products (AGEs), a group of compounds resulting from the non-enzymatic reaction of reducing sugars with the free amino group of proteins, are implicated in diabetic complications. We previously demonstrated that exposure of the pancreatic islet cell line HIT-T 15 to high concentrations of AGEs significantly decreases cell proliferation and insulin secretion, and affects transcription factors regulating insulin gene transcription. The glucagon-like peptide-1 (GLP-1) is an incretin hormone that increases proinsulin biosynthesis, stimulates insulin secretion, and improves pancreatic beta-cell viability. The aim of this work was to investigate the effects of GLP-1 on the function and viability of HIT-T 15 cells cultured with AGEs. HIT-T 15 cells were cultured for 5days in presence of AGEs alone, or supplemented with 10nmol/l GLP-1. Cell viability, insulin secretion, redox balance, and expression of the AGEs receptor (RAGE) were then determined. The results showed that GLP-1 protected beta cell against AGEs-induced cell death preventing both apoptosis and necrosis. Moreover, addition of GLP-1 to the AGEs culture medium restored the redox balance, improved the responsiveness to glucose, and attenuated AGEs-induced RAGE expression. These findings provide evidence that GLP-1 protects beta cells from the dangerous effects of AGEs.

Advanced glycation end-products affect transcription factors regulating insulin gene expression.

Advanced Glycation End-Products (AGEs) are generated by the covalent interaction of reducing sugars with proteins, lipids or nucleic acids. AGEs are implicated in diabetic complications and pancreatic beta-cell dysfunction. We previously demonstrated that exposure of the pancreatic islet cell line HIT-T15 to high concentrations of AGEs leads to a significant decrease of insulin secretion and content. Insulin gene transcription is positively regulated by the beta cell specific transcription factor PDX-1 (Pancreatic and Duodenal Homeobox-1). On the contrary, the forkhead transcription factor FoxO1 inhibits PDX-1 gene transcription. Activity of FoxO1 is regulated by post-translational modifications: phosphorylation deactivates FoxO1, and acetylation prevents FoxO1 ubiquitination. In this work we investigated whether AGEs affect expression and subcellular localization of PDX-1 and FoxO1. HIT-T15 cells were cultured for 5 days in presence of AGEs. Cells were then lysed and processed for subcellular fractionation. We determined intracellular insulin content, then we assessed the expression and subcellular localization of PDX-1, FoxO1, phosphoFoxO1 and acetylFoxO1. As expected intracellular insulin content was lower in HIT-T15 cells cultured with AGEs. The results showed that AGEs decreased expression and nuclear localization of PDX-1, reduced phosphorylation of FoxO1, and increased expression and acetylation of FoxO1. These results suggest that AGEs decrease insulin content unbalancing transcription factors regulating insulin gene expression.

Caveolin-1 is essential for glimepiride-induced insulin secretion in the pancreatic betaTC-6 cell line.

The K(ATP) channels play a pivotal role in the complex mechanism of insulin secretion. K(ATP) channels represent the target of sulphonylureas, a class of drugs widely used in type 2 diabetes to stimulate insulin secretion. We previously showed that caveolin-1 depletion impairs action of the sulphonylurea glimepiride in human endothelial cells. The aim of this work was to investigate the possible role of caveolin-1 in glimepiride-induced insulin secretion. Caveolin-1 was depleted using siRNA method in the pancreatic betaTC-6 cell line. Then stimulation of insulin secretion was performed with different secretagogues (glucose, KCl, and glimepiride). Here, we show that betaTC-6 caveolin-1 depleted cells maintained high rate of insulin secretion after KCl, but not after glucose and glimepiride stimulation. Moreover, we find a direct interaction between caveolin-1 and Kir6.2, one of the K(ATP) channel subunit. These results demonstrate that Cav-1 plays a critical role for glucose and sulfonylurea-stimulated insulin secretion.

Homocysteine, reactive oxygen species and nitric oxide in type 2 diabetes mellitus.

INTRODUCTION: Type 2 diabetes mellitus shows a characteristic altered platelet function that can be due to several mechanisms such as oxidative stress. Hyperhomocysteinemia, considered as a risk factor for various arterial thrombosis, may have a role in generating oxidative damage, even if the pathogenic mechanisms are still not clear. In this report we aimed to determine the role of plasma homocysteine in inducing oxidative stress in type 2 diabetes mellitus. MATERIALS AND METHODS: The study was performed on a group of 34 males with type 2 diabetes and 36 healthy subjects matched for sex and age. Patients and healthy subjects were undergone to laboratory evaluation for plasma homocysteine levels and other metabolic parameters. In both groups of subjects platelet reactive oxygen species, nitric oxide and guanosine 3',5' cyclic monophosphate levels were measured. Moreover the reduced glutathione content in platelets of patients and of healthy subjects was assayed. RESULTS: Plasma homocysteine levels were significantly increased in patients compared with healthy subjects. The basal level of reactive oxygen species was significantly higher in patients than in controls. In addition platelets of patients stimulated with thrombin produced more reactive oxygen species than healthy subjects ones. The nitric oxide, guanosine 3',5' cyclic monophosphate and reduced glutathione content were decreased in platelets of patients. CONCLUSIONS: As homocysteine stimulates oxidative stress and inhibits nitric oxide formation, hyperhomocysteinemia measured in type 2 diabetic patients, promoting platelet hyperactivity, could have a role in the atherogenic effects described in type 2 diabetes.

Rat serum improves rat pseudoislet formation and insulin gene expression.

Rat islet cells in culture are able to form tridimensional aggregates with an architecture and functional activity similar to native islets: pseudoislets. Pseudoislets represent an alternative source for islet transplantation, because their transplant results in a long term allograft acceptance without immunosuppression of the host. Use of pseudoislets has been limited by their reduced yield and by poor reaggregation mass. Since culture conditions have been reported to affect reaggregation, the aim of this study was to evaluate the effects of different concentrations of two sera (Fetal Bovine Serum [FBS] and Rat Serum [RS]) on reaggregation and insulin gene expression in pseudoislets. Islets were isolated from male Lewis rat by means of histopaque gradient centrifugation. The day after islets were disrupted into single cells and cultured in RPMI 1640 5.6 mM glucose with 2%, 5% and 10% solutions of both FBS and RS. Cells spontaneously reaggregated to form pseudoislets. After seven days of culture, pseudoislets were counted and analysed for insulin secretion and insulin gene expression using RT-PCR. Rat serum increased the number of aggregates and their diameters. Insulin gene expression of pseudoislets cultured with RS showed a ten fold increase in comparison to those cultured with FBS. These data show that the culture medium supplemented with RS improves total reaggregate volume and increases insulin gene expression. With the perspective of pseudoislets' use in transplantation RS is better indicated than FBS for the production of rat pseudoislets.

Reduced glucose effectiveness as a feature of glucose intolerance: evidence in elderly type-2 diabetic subjects.

One of the factors determining glucose tolerance is glucose disappearance independent from the dynamic insulin (glucose effectiveness); the debate on its role in the development of Type-2 diabetes is still open. The aim of the present study was to evaluate insulin delivery, insulin sensitivity (SI), and glucose effectiveness (SG) in a group of elderly Type-2 diabetic patients (D, 4/6 F/M, age 67 +/- 2 years, 64 +/- 2 kg, BMI 23.8 +/- 0.5 kg/m2), compared to young controls (C, 4/6 F/M, 25 +/- 2 years, 72 +/- 4 kg, 23.7 +/- 1.1 kg/m2) and elderly controls (E, 2/4 F/M, 73 +/- 3 years, 63 +/- 4 kg, 23.1 +/- 0.5 kg/m2). We performed oral (OGTT) and intravenous (FSIGT) glucose tolerance tests. The OGTT showed that C and E were normotolerant, while D had a markedly reduced glucose tolerance. This was also confirmed in the FSIGT where the glucose tolerance index (KG) was 0.6 +/- 0.1% min-1 in D vs 1.8 +/- 0.2 in C and 1.5 +/- 0.2 in E (p < 0.0002). Total insulin area of D and the overall insulin delivery were not different from those of the control groups. The early phase area was instead significantly reduced (0.19 +/- 0.02 mU min/mL vs 0.61 +/- 0.06 of C and 0.46 +/- 0.06 of E, p < 0.001) given the reduction in the dynamic first-phase insulin delivery (0.86 +/- 0.17 min(microU/mL)/(mg/dL) vs 3.95 +/- 0.61 in C (p < 0.005) and 2.61 +/- 0.66 (p < 0.001) in E). SI of D was 3.4 +/- 0.4 10(-4) min-1/(microU/mL), not different from that of C (4.7 +/- 0.6) and E (3.5 +/- 0.2). This study showed a marked difference between SG of D and that of both control groups [0.010 +/- 0.001 min-1 vs 0.026 +/- 0.004 (p < 0.001) of C and 0.020 +/- 0.003 (p < 0.002) of E], mostly due to the zero-insulin component GEZI which was 0.006 +/- 0.001 in D vs 0.021 +/- 0.004 in C and 0.016 +/- 0.003 in E (p < 0.003). In the elderly groups, when taken together, SG exhibited a positive correlation with the area under insulin concentration during the early phase and with KG (r = 0.69, p = 0.0032 and r = 0.90, p = 0.0001, respectively), demonstrating the importance of the first-phase insulin delivery in modulating glucose effectiveness and glucose tolerance.

Water distribution in insulin-dependent diabetes mellitus in various states of metabolic control.

Alterations in water compartments have been described in insulin-dependent diabetes mellitus (IDDM). Both insulin and lack of natriuretic counteracting response lead to water expansion, while hyperglycemica-induced osmotic diuresis leads to water depletion. Both total body water and water distribution in the extra-intracellular space, as well as their relationships to metabolic control, were investigated in 15 controls (30.1 +/- 1.4 years) and in 26 IDDM patients (31.3 +/- 1.6, diabetes duration 11.3 +/- 1.4 years) who were neither hypertensive nor proteinuric. The amounts of total body water (TBW) and extracellular water (ECW) were predicted by impedance measurements at 100 KHz and at 1 KHz. The amount of intracellular water (ICW) was computed as the difference between the two. Water distribution was estimated by measuring the ratio between low- and high-frequency impedance and by computing the ratio between ECW and ICW. The IDDM patients were divided into four groups on the basis of reference HbA(lc) mean and SD: A < or = mean + 2 SD < B < or = mean + 4 SD < C < or = mean +6SD < D. The groups were comparable with sodium intake, insulin dosage, fasting glycemia and laboratory hydration markers. As compared to controls, impedance values at 1, 5, 10, 50 and 100KHz were significantly lower in diabetic patients and the difference within group D increased as the frequency increased: -3.9% at 1 KHz, -10.1% at 100 KHz. As compared to controls, groups A, B and C showed higher TBW, ECW and ICW while water distribution was normal, and group D showed higher TBW and ICW but normal ECW and a different water distribution. In all IDDM patients, HbA(lc) correlated with ECW (r = -0.49) and distribution ratios (r = 0.42, impedance; r = 0.40, ECW/ICW ratio). These observations suggest that good or moderate long-term control IDDM patients have proportionately normal distributions of ECW and ICW excess. However, water excess in poor control IDDM patients was only found in the ICW space.

An instrument for real-time spectral estimation of heart rate variability signals.

A Digital Signal Processor (DSP)-based instrument is proposed for estimating and displaying the Heart Rate Variability (HRV) spectrum in real-time. It consists of an intelligent module which is properly interfaced to an IBM PC and whose operations are independent from the computer's other tasks. In this way, the simultaneous recording of the ECG sequence, needed for the more complete off-line analysis, can be performed by the same host. The employed hybrid spectral estimator (in which a classical FFT analysis follows the autoregressive extrapolation of data) appears to be the most apt for the present fixed point arithmetics implementation. The reliability of the instrument and its accuracy are checked both with suitable test signals and by comparison with the results obtained through off-line analysis of the same ECG tracks. The instrument is presently used for cardiovascular investigations, in particular for quickly picking patients with cardiac autonomic neuropathy (CAN) out of a population of diabetic subjects.

Impairment of central motor conduction in diabetic patients.

In 70 patients with diabetes mellitus (DM) we recorded the motor evoked potentials (MEPs) following magnetic stimulation of the motor cortex and spinal roots. Central motor conduction time (CMCT) was determined as the difference between MEP latencies after cortical and spinal stimulation. The mean CMCTs for the biceps, thenar and tibialis anterior muscles were prolonged in the DM group, as compared to normal controls, and 21 patients exceeded the CMCT upper confidence limit for at least one muscle. CMCT changes and peripheral conduction velocity abnormalities occurred independently and were related to different clinical parameters. We conclude that a subclinical impairment of central motor conduction is present in 30% of DM patients, independently from the occurrence of a diabetic peripheral neuropathy and possibly reflecting different pathophysiological mechanisms.

Isolation of pig pancreatic islets by a new method with hydraulic shaking: preliminary report.

The limited availability of human pancreas represents a serious problem in islet transplantation. In the past few years many efforts have been made to isolate pancreatic islets from large mammals in order to achieve valid and reproducible isolation methods. For several reasons swine may be considered an ideal source of islet tissue because of the similarity between human and porcine insulin and because of the easy availability of pig pancreata. Some papers have been published recently on this topic with good results. However, some problems, such as islet dissociation into single cells after collagenase digestion, are not completely solved. In this article, an automated method involving a hydraulic shaking system is described for islet isolation from the pig pancreas, developed in our laboratory and derived from Ricordi's model.

Comparison of the effects of carvedilol and nifedipine in patients with essential hypertension and non-insulin-dependent diabetes mellitus.

The safety and tolerability of carvedilol, a new antihypertensive agent with the combined pharmacological activities of beta-blockade and vasodilation, and of nifedipine were investigated in patients with essential hypertension and non-insulin-dependent (type II) diabetes mellitus. Twenty patients were openly randomized to receive 25 mg carvedilol once daily (five men and five women; mean age, 63 years) or 10 mg nifedipine t.i.d. (three men and seven women; mean age, 64 years) for a period of 4 weeks. Baseline mean sitting blood pressures were 168/98 and 169/95 mm Hg in the carvedilol and nifedipine groups, respectively. Baseline mean areas under the curve (AUC) of the intravenous glucose tolerance test (IVGTT) for the carvedilol and nifedipine groups were 6,136 +/- 1,195 and 6,287 +/- 1,228 mg/dl/min, respectively. Demographic and efficacy variables were not statistically different between treatment groups. After 4 weeks of therapy, mean sitting blood pressure was significantly (p less than 0.02) reduced to 144/91 mm Hg in the carvedilol group and to 149/87 mm Hg in the nifedipine group. Week 4 IVGTT AUC values of 5,735 +/- 1,464 mg/dl/min in the carvedilol group and 5,988 +/- 993 mg/dl/min in the nifedipine group, representing mean reductions of 6.14% and 3.17%, respectively, were not statistically different from baseline. Both treatments were well tolerated. No patient experienced adverse events in the carvedilol treatment group, whereas two patients in the nifedipine group reported episodes of headache (one patient) and palpitations (one patient); each episode was mild in severity and considered to be related to study medication.(ABSTRACT TRUNCATED AT 250 WORDS)

[Insulin secretion and hepatic extraction in juvenile non-insulin-dependent diabetes]. / Secrezione insulinica ed estrazione epatica nel diabete non insulino dipendente giovanile.

Beta-cell secretion is still a point of controversy. As the liver is the major site of insulin metabolism, evaluation of hepatic insulin extraction is crucial for correct measurement of beta-cell secretion. Methods for calculating the secretion and hepatic extraction of insulin indirectly from peripheral C-peptide concentration have been proposed by some investigators. To characterize the low insulin response of a group of young non-insulin-dependent diabetics we evaluated secretion and hepatic insulin extraction during an oral glucose tolerance test by peripheral IRCP determination and IRCP/IRI molar ratio. Our data show that in this population of young non-insulin-dependent diabetics, the low peripheral insulin response to an oral glucose challenge is a possible consequence of diminished beta-cell secretion, as hepatic insulin extraction is at near normal value.