Francesco Purrello

Functional and morphological alterations of mitochondria in pancreatic beta cells from type 2 diabetic patients

Aims/hypothesisLittle information is available on the insulin release properties of pancreatic islets isolated from type 2 diabetic subjects. Since mitochondria represent the site where important metabolites that regulate insulin secretion are generated, we studied insulin release as well as mitochondrial function and morphology directly in pancreatic islets isolated from type 2 diabetic patients.MethodsIslets were prepared by collagenase digestion and density gradient purification, and insulin secretion in response to glucose and arginine was assessed by the batch incubation method. Adenine nucleotides, mitochondrial membrane potential, the expression of UCP-2, complex I and complex V of the respiratory chain, and nitrotyrosine levels were evaluated and correlated with insulin secretion.ResultsCompared to control islets, diabetic islets showed reduced insulin secretion in response to glucose, and this defect was associated with lower ATP levels, a lower ATP/ADP ratio and impaired hyperpolarization of the mitochondrial membrane. Increased protein expression of UCP-2, complex I and complex V of the respiratory chain, and a higher level of nitrotyrosine were also found in type 2 diabetic islets. Morphology studies showed that control and diabetic beta cells had a similar number of mitochondria; however, mitochondrial density volume was significantly higher in type 2 diabetic beta cells.Conclusions/interpretationIn pancreatic beta cells from type 2 diabetic subjects, the impaired secretory response to glucose is associated with a marked alteration of mitochondrial function and morphology. In particular, UCP-2 expression is increased (probably due to a condition of fuel overload), which leads to lower ATP, decreased ATP/ADP ratio, with consequent reduction of insulin release.

An overview of pancreatic beta-cell defects in human type 2 diabetes: Implications for treatment

Type 2 diabetes is the most common form of diabetes in humans. It results from a combination of factors that impair beta-cell function and tissue insulin sensitivity. However, growing evidence is showing that the beta-cell is central to the development and progression of this form of diabetes. Reduced islet and/or insulin-containing cell mass or volume in Type 2 diabetes has been reported by several authors. Furthermore, studies with isolated Type 2 diabetic islets have consistently shown both quantitative and qualitative defects of glucose-stimulated insulin secretion. The impact of genotype in affecting beta-cell function and survival is a very fast growing field or research, and several gene polymorphisms have been associated with this form of diabetes. Among acquired factors, glucotoxicity, lipotoxicity and altered IAPP processing are likely to play an important role. Interestingly, however, pharmacological intervention can improve several defects of Type 2 diabetes islet cells in vitro, suggesting that progression of the disease might not be relentless.

Adrenocortical dysfunction in liver disease: A systematic review

In patients with cirrhosis, adrenal insufficiency (AI) is reported during sepsis and septic shock and is associated with increased mortality. Consequently, the term “hepato‐adrenal syndrome” was proposed. Some studies have shown that AI is frequent in stable cirrhosis as well as in cirrhosis associated with decompensation other than sepsis, such as bleeding and ascites. Moreover, other studies showed a high prevalence in liver transplant recipients immediately after, or some time after, liver transplantation. The effect of corticosteroid therapy in critically ill patients with liver disease has been evaluated in some studies, but the results remain controversial. The 250‐μg adreno‐cortico‐tropic‐hormone stimulation test to diagnose AI in critically ill adult patients is recommended by an international task force. However, in liver disease, there is no consensus on the appropriate tests and normal values to assess adrenal function; thus, standardization of normal ranges and methodology is needed. Serum total cortisol assays overestimate AI in patients with cirrhosis, so that direct free cortisol measurement or its surrogates may be useful measurements to define AI, but further studies are needed to clarify this. In addition, the mechanisms by which liver disease leads to adrenal dysfunction are not sufficiently documented. This review evaluates published data regarding adrenal function in patients with liver disease, with a particular focus on the potential limitations of these studies as well as suggestions for future studies. (HEPATOLOGY 2012)

Safety of Type 2 Diabetes Treatment With Repaglinide Compared With Glibenclamide in Elderly People: A randomized, open-label, two-period, cross-over trial

The incidence of type 2 diabetes increases with age (1), and elderly people with this disease may be particularly susceptible to hypoglycemia due to long-acting oral antidiabetic drugs (OADs). The American Geriatric Society clinical guidelines on type 2 diabetes treatment in elderly people report that short-acting hypoglycemic agents are preferable to longer-acting agents (chlorpropramide), which are associated with increased risk of hypoglycemia (2). Repaglinide is an insulin secretagogue with a rapid onset and relatively short duration of action (3,4). Several studies have shown repaglinide to be a safe and effective treatment for type 2 diabetes (5–9). However, few data are available on its use in elderly patients and, in particular, on the incidence of hypoglycemic events. The present study assessed the safety of repaglinide versus glibenclamide in this population, in terms of hypoglycemia and adverse events. This was a 24-week, randomized, open-label, two-period, cross-over comparison between mealtime repaglinide and twice-daily glibenclamide. Patients ( n = 90) were aged ≥65 years and had been previously treated with diet or OADs (mean age 74.6 years, HbA1c [A1C] 7.9%). A subgroup of 37 patients aged ≥75 years was evaluated separately. After screening, previous OAD treatment was discontinued and patients were randomized to first receive either repaglinide (Novonorm; …

Effects of High Glucose on Insulin Secretion by Isolated Rat Islets and Purified β-Cells and Possible Role of Glycosylation

We investigated the effect of 24 h of exposure to various glucose concentrations on insulin secretion by isolated rat pancreatic islets and purified rat β-cells. Compared with islets cultured with standard medium (5.5 mM glucose), islets cultured with 16.7 mM glucose showed a higher basal insulin release (means ± SE, 3.0 ± 0.5 vs. 0.7 ± 0.2%, n = 8, P < .005) and reduced glucose-stimulated insulin secretion (2.4 ± 0.3 vs. 5.8 ± 0.4%, n = 8, P < .005). Similar results were also obtained with purified β-cells. The effect of high glucose was time dependent (present after 12 h, maximal after 24 h) and reversible: when islets cultured with high glucose were transferred to standard medium, normal responsiveness to glucose was restored within 8 h and normal basal release within 24 h. Mannitol, 3-O-methylglucose, and 2-deoxyglucose were not able to mimic the effects of glucose. Islets or purified β-cells cultured in the presence of high glucose had a normal response when stimulated with glyburide, dibutyryl cyclic AMP, and isobutylmethylxanthine. Tunicamycin, an inhibitor of N-terminal glycosylation, prevented glucose-induced desensitization when added during 24 h of islet culture with 16.7 mM glucose. Swainsonine, another agent that influences glycosylation, had a similar effect. Our study indicates 1) that 24 h of exposure to high glucose induces a specific and reversible impairment of insulin secretion in response to glucose, 2) that this is a direct effect of glucose on β-cells, and 3) that islet glucose metabolism and glycosylation processes may play a critical role in determining glucose desensitization.

Cellular and molecular effects of protons: Apoptosis induction and potential implications for cancer therapy

Due to their ballistic precision, apoptosis induction by protons could be a strategy to specifically eliminate neoplastic cells. To characterize the cellular and molecular effects of these hadrons, we performed dose-response and time-course experiments by exposing different cell lines (PC3, Ca301D, MCF7) to increasing doses of protons and examining them with FACS, RT-PCR, and electron spin resonance (ESR). Irradiation with a dose of 10 Gy of a 26,7 Mev proton beam altered cell structures such as membranes, caused DNA double strand breaks, and significantly increased intracellular levels of hydroxyl ions, are active oxygen species (ROS). This modified the transcriptome of irradiated cells, activated the mitochondrial (intrinsic) pathway of apoptosis, and resulted in cycle arrest at the G2/M boundary. The number of necrotic cells within the irradiated cell population did not significantly increase with respect to the controls. The effects of irradiation with 20 Gy were qualitatively as well as quantitatively similar, but exposure to 40 Gy caused massive necrosis. Similar experiments with photons demonstrated that they induce apoptosis in a significantly lower number of cells and in a temporally delayed manner. These data advance our knowledge on the cellular and molecular effects of proton irradiation and could be useful for improving current hadrontherapy protocols.

Assessment of adrenocortical reserve in stable patients with cirrhosis

BACKGROUND & AIMS Adrenal insufficiency (AI) is reported in critically ill patients with cirrhosis and is associated with increased mortality. It is unclear if AI is an underlying condition or triggered by critical events (e.g. sepsis). We investigated AI in cirrhosis without infection or hemodynamic instability. METHODS A total of 101 consecutive patients with cirrhosis were studied. AI was defined by a total serum cortisol (TC) <18 μg/dl at 20 or 30 min after injection of 1 μg of tetracosactrin. Transcortin, calculated free cortisol (cFC), and free cortisol index (FCI) were assessed in a subgroup of 41 patients, with FCI>12 representing normal adrenal function. RESULTS AI was present in 38 patients (38%). Child score (median, 10 vs 7, p<0.0001), MELD score (median, 17 vs 12, p<0.0001), ascites (68% vs 37%, p<0.01), basal TC (median,7.6 vs 14.9 μg/dl, p<0.001), albumin (28 ± 0.8 vs 33 ± 0.7 g/L, p<0.0001), INR (median, 1.6 vs 1.2, p<0.0001), total bilirubin (median, 51 vs 31 μmol/L, p<0.05), total cholesterol (median, 120 vs 142, p<0.05), and LDL (median, 76 vs 81, p<0.05) were significantly different between those with and without AI. ROC curves showed a basal TC ≤ 12.8 μg/dl to be a cut-off value closely associated with AI. The cFC was significantly related to TC for baseline values (R=0.94, p<0.0001), peak values (R=0.90, p<0.0001), and delta values (R=0.95, p<0.0001), in patients with and without AI. However, no patient had a FCI<12. CONCLUSIONS AI defined by an abnormal response to 1 μg tetracosactrin is frequent in stable patients with cirrhosis, in the absence of infections or hemodynamic instability and is related to the severity of liver disease. However, evaluation of the true incidence of AI should comprise direct assays of free cortisol. Clinical consequences of AI need to be explored.

Type 2 diabetes susceptibility gene expression in normal or diabetic sorted human alpha and beta cells: correlations with age or BMI of islet donors

Background Genome-wide association studies have identified susceptibility genes for development of type 2 diabetes. We aimed to examine whether a subset of these (comprising FTO, IDE, KCNJ11, PPARG and TCF7L2) were transcriptionally restricted to or enriched in human beta cells by sorting islet cells into alpha and beta – specific fractions. We also aimed to correlate expression of these transcripts in both alpha and beta cell types with phenotypic traits of the islet donors and to compare diabetic and non-diabetic cells. Methodology/Principal Findings Islet cells were sorted using a previously published method and RNA was extracted, reverse transcribed and used as the template for quantitative PCR. Sorted cells were also analysed for insulin and glucagon immunostaining and insulin secretion from the beta cells as well as insulin, glucagon and GLP-1 content. All five genes were expressed in both alpha and beta cells, with significant enrichment of KCNJ11 in the beta cells and of TCF7L2 in the alpha cells. The ratio of KCNJ11 in beta to alpha cells was negatively correlated with BMI, while KCNJ11 expression in alpha cells was negatively correlated with age but not associated with BMI. Beta cell expression of glucagon, TCF7L2 and IDE was increased in cells from islets that had spent more time in culture prior to cell sorting. In beta cells, KCNJ11, FTO and insulin were positively correlated with each other. Diabetic alpha and beta cells had decreased expression of insulin, glucagon and FTO. Conclusions/Significance This study has identified novel patterns of expression of type 2 diabetes susceptibility genes within sorted islet cells and suggested interactions of gene expression with age or BMI of the islet donors. However, expression of these genes in islets is less associated with BMI than has been found for other tissues.

Early, but Not Advanced, Glomerulopathy Is Reversed by Pancreatic Islet Transplants in Experimental Diabetic Rats: Correlation With Glomerular Extracellular Matrix mRNA Levels

In this study, we investigated 1) whether long-term restoration of euglycemia by means of pancreatic islet transplants is capable of preventing and/or reversing renal functional and structural alterations in an experimental model of insulin-deficient diabetes, and 2) whether changes in extracellular matrix (ECM) and cell turnover at the glomerular level and biochemical abnormalities associated with hyperglycemia correlate with the renal outcome after transplantation. Male Lewis rats, rendered diabetic by intravenous injection of streptozotocin, underwent homologous islet transplantation via the portal vein at 2 weeks (study A), at 4 months (study B), and at 8 months (study C) after the induction of diabetes and killed 12 months after transplantation in study A and 4 months after transplantation in studies B and C. Age-matched nondiabetic and untreated diabetic rats were used as control animals and were studied at 4, 8, and 12 months. In the untreated diabetic animals, metabolic derangement was associated with increased erythrocyte polyol and fructose levels, tail-tendon content of advanced glycation end products (AGEs), total proteinuria, albuminuria, kidney weight, and mean glomerular volume as well as with marked glomerular and extraglomerular lesions. Glomerular gene expression for the ECM components fibronectin and collagen IV and for TGF-β was also increased, whereas glomerular cell proliferation was unaffected by diabetes. In study A, changes in renal function and structure observed in diabetic rats at 12 months were completely prevented by successful islet transplants. In study B, all functional and structural abnormalities detected in diabetic rats at 4 months of disease duration were virtually reversed by 4 months of euglycemia in transplanted animals, whereas they progressed further in untreated diabetic rats. In study C, the course of functional and structural changes observed in untreated diabetic rats was not reversed by islet transplantation. Likewise, tissue AGE accumulation and particularly upregulation of glomerular ECM and transforming growth factor (TGF)-β gene expression, which are believed to play a role in the pathogenesis of altered renal function and structure in diabetes, were normalized in transplanted rats from study A and study B, but not in those from study C. These experiments show that restoration of euglycemia by islet transplants is capable of preventing experimental diabetic glomerulopathy and reversing early changes in renal function and structure induced by diabetes. In a later phase of the disease, when glomerular matrix gene expression becomes independent of hyperglycemia, possibly because of the persistent increase in tissue AGE accumulation, metabolic control is not capable of reversing renal abnormalities.

Glucose Modulates Glucose Transporter Affinity, Glucokinase Activity, and Secretory Response in Rat Pancreatic β-Cells

Pancreatic islets were cultured for 24 h in medium containing either low (1.4), normal (5.5), or high (16.7 mM) glucose, and then insulin secretion was measured at the end of 1 h incubation at 37°C. Insulin release in the absence of glucose was 64 ± 20,152 ± 11, and 284 ± 30 pg · islet−1 · h−1 (mean ± SE, n = 6, G1.4 and G16.7 vs. G.5.5, P < 0.05) and the response to 22 mM glucose stimulation was 640 ± 136, 2460 ± 276, and 1890 ± 172 pg · islet−1 · h−1, respectively (n = 6, G1.4 vs. G5.5, P < 0.01, G16.7 vs. G5.5, P = 0.065). The 50% maximal response of insulin secretion (increment over baseline) was reached at an average glucose concentration of 9.9 ± 0.7 mM in islets preexposed to G5.5, and at glucose 13.3 ± 0.9 and 4.8 ± 0.4 mM (P < 0.05 in respect to G5.5) in islets preexposed to G1.4 and G16.7, respectively. To investigate the molecular mechanism responsible for this altered glucose sensitivity, we measured, in parallel experiments, the kinetic characteristics of glucose transport, glucokinase, and glucose utilization. Glucose transport was measured by evaluating 3-O-methylglucose uptake. The apparent Km of the low-affinity transporter (GLUT2) was 16.6 ± 2.4 mM in isolated pancreatic cells cultured at 5.5 mM glucose. In cells cultured at both low (1.4 mM) or high (16.7 mM) glucose concentrations, a significant change in the apparent Km of this glucose transporter function was observed (24.4 ± 2.9 and 7.1 ± 0.6 mM, n = 5, P < 0.05 and < 0.01, respectively), with no change in the Vmax of the uptake. Under the same experimental conditions a concomitant change in glucokinase activity was observed: the enzyme Vmax was 4.9 ± 0.32, 8.7 ± 0.79, and 15.8 ± 0.98 μmol · μg DNA−1 · h−1 in islets From the Institute of Internal Medicine, Metabolism and Endocrinologyexposed to either 1.4, 5.5, or 16.7 mM glucose, respectively (mean ± SE, n = 5, G1.4 and G16.7 vs. G.5.5, P < 0.05), with no significant change in the enzyme Km. In control islets glucose utilization, measured by 3H20 production from [5-3H]glucose, had a Km of 8.0 ± 1.7 mM and a Vmax of 8.9 ± 0.41 nmol · μg DNA−1 · 2 h−1. In islets exposed to either G1.4 or G16.7, the glucose utilization Vmax was decreased and increased (5.3 ± 0.56 and 13.8 ± 0.98 nmol · μg DNA−1 · 2 h−1 n = 5, P < 0.05), parallel with the changes observed in glucokinase activity. Moreover, the apparent glucose utilization Km was increased in G1.4 preexposed islets (15.6 ± 1.3 mM) and decreased in G16.7 preexposed islets (3.7 ± 0.9 mM), parallel with the changes observed in the glucose transport Km. These studies indicate that in vitro the sensitivity and responsiveness of pancreatic islets to glucose may be regulated by the ambient glucose concentrations. They support the concept that glucokinase plays a pivotal role in regulating glucose metabolism Vmax, and, therefore, the β-cell responsiveness to glucose. In addition, they also indicate that changes in the affinity (Km) of the glucose transporter are associated to changes in the Km of glucose utilization, thus suggesting a possible role of glucose transport in determining the β-cell sensitivity to glucose.