Beta cell response to a mixed meal in nigerian patients with type 2 diabetes

ABSTRACT: BACKGROUND: The pathophysiology of type2 diabetes involves both insulin resistance and poor beta cell function. Studies have been done in several populations to assess the relative importance of these mechanisms in individual patients. In our environment studies to assess beta cell function have been done with glucagon stimulation or an oral glucose tolerance test. This study was done to assess the response of the beta cell to a standardized mixed meal and its relationship with glycaemic control in patients with type2 diabetes. METHODS: Ninety patients with type 2 diabetes were recruited into the study. Weight, height, body mass index and waist circumference were measured. Blood samples were analysed for fasting plasma glucose (FPG) and fasting C peptide (FCP) and glycated haemoglobin (HbA1c). Patients were given their usual drugs for management of their diabetes and then served with a standard meal calculated to contain 50 g of carbohydrate, made up of 53 % carbohydrate, 17 % of protein and 30 % of lipids, providing 500 kcal. Blood samples 2 hours after the start of the meal were analysed for postprandial glucose (PPG) and postprandial C peptide (PCP). Fasting (M0) and postprandial beta cell responsiveness (M1) were calculated. RESULTS: The mean FPG and PPG were 7.51+/ 3.39 mmol/l and 11.02+/4.03 mmol/l respectively while the mean glycated haemoglobin (HbA1c) was 9.0+/2.5 %. The mean fasting C peptide was 1.44+/1.80ug/ml. Many of the patients (56.7 %) had low FCP levels. The mean postprandial C peptide was 4.0+/2.8 ng/ml. There were significant correlations between M1, HbA1c and PPG (p = 0.015, 0.024, 0.001 respectively) and also between M0, HbA1c, PPG and FPG (p = 0.001, 0.002, 0.001). HbA1c decreased across increasing tertiles of M0 (p < 0.001) and also M1 (p = 0.002). In step-wise linear regression analysis, M0 and M1 significantly predicted HbA1c. CONCLUSIONS: Many of the patients had low C peptide levels with poor beta cell response to the meal. The patients had poor glycaemic control and poor beta cell function. Both fasting and postprandial beta cell responsiveness were significant determinants of blood glucose and glycated haemoglobin levels. It is likely that putting these patients on insulin may have led to better glycaemic control in them.     

Sensitization to alloxan-induced diabetes and pancreatic cell apoptosis in acatalasemic mice

Human acatalasemia may be a risk factor for the development of diabetes mellitus. However, the mechanism by which diabetes is induced is still poorly understood. The impact of catalase deficiency on the onset of diabetes has been studied in homozygous acatalasemic mutant mice or control wild-type mice by intraperitoneal injection of diabetogenic alloxan. The incidence of diabetes was higher in acatalasemic mice treated with a high dose (180 mg/kg body weight) of alloxan. A higher dose of alloxan accelerated severe atrophy of pancreatic islets and induced pancreatic β cell apoptosis in acatalasemic mice in comparison to wild-type mice. Catalase activity remained low in the acatalasemic pancreas without the significant compensatory up-regulation of glutathione peroxidase or superoxide dismutase. Furthermore, daily intraperitoneal injection of angiotensin II type 1 (AT1) receptor antagonist telmisartan (0.1 mg/kg body weight) prevented the development of alloxan-induced hyperglycemia in acatalasemic mice. This study suggests that catalase plays a crucial role in the defense against oxidative-stress-mediated pancreatic β cell death in an alloxan-induced diabetes mouse model. Treatment with telmisartan may prevent the onset of alloxan-induced diabetes even under acatalasemic conditions.     

Beta blockade induces apoptosis in cultured capillary endothelial cells

Continuous beta blockade stimulates deposition of collagen in the pulmonary alveolar interstitium of adult rats. It also causes changes to the capillary endothelial cell compartment reminiscent of programmed cell death. To test whether beta blockade results in endothelial cell apoptosis, cultures of capillary endothelial cells were treated with both a wide-spectrum beta blocker and a beta-2-specific antagonist. Apoptosis was measured in these cultures using both terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling and annexin-V assays. Both forms of beta blockade stimulated programmed cell death in these cultures. To test whether the apoptotic effect of beta blockade was related to interstitial collagen deposition, capillary endothelial cells were cocultured with beta-blocked pulmonary fibroblast monolayers. Cocultured endothelial cells were substantially protected from apoptosis after beta blockade; coculture over plain tissue culture plastic or over exogenous collagen films had no effect on programmed cell death in endothelial cells. These results suggest that both pulmonary endothelial and interstitial cells are vulnerable to injury from beta blockade but that paracrine interactions between these cells may protect the peripheral lung from substantive damage.     

Beta cell contact and insulin release

Insulin secretion by intact islets, dispersed islet cells and dispersed cells allowed to reaggregate was compared in perifusion. Although single cells and aggregates showed basal insulin secretion and a prompt response to glucose challenge, basal secretion, peak insulin secretion and total insulin secretion during a 60 minute stimulation were profoundly less than those activities of intact islets. These results suggest that dispersed beta cells are responsive to glucose as a secretagogue, but the magnitude of the response is greatly diminished and not restored by simple cell contact.     

Small molecular weight G-protein, H-Ras, and retinal endothelial cell apoptosis in diabetes

We have demonstrated that the expressions of small molecular weight G-protein, H-Ras, and its effector protein, Raf-1, are increased in the retina in diabetes, and the specific inhibitors of Ras function inhibit glucose-induced apoptosis of retinal capillary cells. This study is to examine the contributory roles for H-Ras in glucose-induced apoptosis of retinal endothelial cells by genetic manipulation of functionally active H-Ras levels. Bovine retinal endothelial cells were transfected with the plasmids of either wild type (WT), constitutively active (V12) or dominant-negative (N17) H-Ras. Glucose-induced increase in apoptosis, nitric oxide (NO) levels and activation of NF-κB and caspase-3 were determined in these genetically manipulated cells. Exposure of bovine retinal endothelial cells to 20 mM glucose significantly increased H-Ras activation as determined by Raf-1 binding assay. Overexpression of V12 in the endothelial cells further increased their glucose-induced apoptosis by 40%, NO levels by about 50%, and activated NF-κB and caspase-3 by about 30–40% compared to the untransfected cells incubated in 20 mM glucose. In contrast, overexpression of the inactive mutant, N17, inhibited glucose-mediated increases in apoptotic cell death, NO levels and NF-κB and caspase-3 activation; the values were significantly different (p < 0.02) compared to those obtained from the untransfected cells incubated under similar conditions. Our findings demonstrate that H-Ras activation is important in the activation of the specific signaling events leading to the accelerated retinal capillary cell apoptosis in hyperglycemic conditions, suggesting the possible use of H-Ras inhibitors to inhibit the pathogenesis of diabetic retinopathy.     

Two autoimmune diabetes loci influencing T cell apoptosis control susceptibility to experimental autoimmune myocarditis

The pathogenesis of immune-mediated myocarditis depends on genetic and environmental factors. To study the genetic mechanisms, we have developed a model of experimental autoimmune myocarditis in the A.SW mouse. Here we provide evidence that loci on murine chromosome 6, and possibly chromosome 1, are involved in regulating susceptibility. Moreover, these loci overlap with loci implicated in other autoimmune diseases including diabetes in the NOD mouse. These two loci also regulate apoptosis in thymocytes as well as peripheral T cells in the NOD mouse, and we report further that A.SW mice demonstrate the same characteristics in apoptosis. These results suggest that common pathogenetic mechanisms involving apoptosis of both thymic and peripheral T cells are shared by multiple autoimmune diseases.     

Beta4 integrin is involved in statin-induced endothelial cell death

HMG-CoA reductase inhibitors (statins) have been shown to inhibit angiogenesis. The molecular mechanism mediating the anti-endothelial activities of statins remains unclear. The present study demonstrated that the antiangiogenic effect of atorvastatin (ATV) was associated with endothelial death. Molecular profiling data identified a 29-fold upregulation of beta4 integrin mRNA. Western blot and flow cytometry confirmed robust increases of total and cell-surface beta4 integrin. Blockage of beta4 integrin activity by antagonizing antibody abrogated ATV-induced endothelial death. The endothelial death and beta4 integrin upregulation by ATV could be reversed by intermediate metabilites of the HMG-CoA reductase pathway mevalonate or GGPP, but not by FPP, suggesting that these effects were results of specific inhibition of the pathway. These data indicate that the HMG-CoA reductase might represent an important survival pathway in angiogenic endothelial cells and thus, a potential target for antiangiogenic therapy.     

Endoplasmic reticulum stress-induced apoptosis and auto-immunity in diabetes

Increasing evidence suggests that stress signaling pathways emanating from the endoplasmic reticulum (ER) are important to the pathogenesis of both type 1 and type 2 diabetes. Recent observations indicate that ER stress signaling participates in maintaining the ER homeostasis of pancreatic beta-cells. Either a high level of ER stress or defective ER stress signaling in beta-cells may cause an imbalance in ER homeostasis and lead to beta-cell apoptosis and autoimmune response. In addition, it has been suggested that ER stress attributes to insulin resistance in patients with type 2 diabetes. It is necessary to study the relationship between ER stress and diabetes in order to develop new therapeutic approaches to diabetes based on drugs that block the ER stress-mediated cell-death pathway and insulin resistance.     

High levels of filamentous actin and apoptosis correlate with mast cell refractoriness under alloxan-evoked diabetes

Mast cell number and reactivity were shown to be down-regulated under diabetic conditions. Since the balance between globular and filamentous actin plays a pivotal role in the activity of secretory cells, we investigated whether an imbalance in that system could underlie the hyporesponsiveness of mast cells in diabetes. The apoptotic state was also evaluated. By means of rhodamine/phalloidine staining of F-actin, we noted that diabetic mast cells exhibited an increase in fluorescence intensity and reduction in cellular size, when compared with cells from normal animals, in parallel with elevation in the percentage of cells developing apoptosis. The levels of Bax, a pro-apoptotic member of Bcl-2 family, appeared increased at baseline in mast cells from diabetic rats compared with normal cells. These phenomena correlated with reduction in histamine and PGD2 release following antigen challenge in vitro. The steroid antagonist RU 486 abolished the reduction of histamine secretion from diabetic mast cells. We conclude that hyporesponsiveness of mast cells noted in diabetes may be accounted for by reduction in actin filament plasticity, in clear association with the rise in the percentage of cells undergoing apoptosis. In addition, the refractoriness of diabetic mast cells to antigen in vitro seems to be dependent on glucocorticoids.     

Beta cell cytoprotection using lentiviral vector-based iNOS-specific shRNA delivery

Cytokine-induced beta cell pathophysiology is characterised by the induction of iNOS expression. Inhibition of iNOS expression protects beta cells from cytokine-mediated destruction. The development of vector-based shRNA strategies capable of stably suppressing iNOS expression may provide a novel platform to protect beta cells from cytokine toxicity. In this report the utility of lentiviral shRNA vectors to silence iNOS expression was evaluated with respect to insulinoma cell viability, the induction of iNOS expression and the accumulation of nitrite in a cytokine-induced beta cell toxicity model. Here, we report for the first time on the use of lentiviral vector-based shRNA delivery to efficiently suppress the IL-1beta-mediated induction of iNOS expression, the accumulation of nitrite and provide significant protection against the cytotoxic effects of IL-1beta exposure. Moreover, non-specific knockdown of endogenous beta cell nNOS did not occur.     

Regulating apoptosis in mammalian cell cultures

Cell culture technology has become a widely accepted method used to derive therapeutic and diagnostic protein products. Mammalian cells adapted to grow in bioreactors now play an integral role in the development of these biologicals. A major limiting factor determining the output efficiency of mammalian cell cultures however, is apoptosis or programmed cell death. Methods to delay apoptosis and increase the longevity of cell cultures can lead to more economical processes. Researchers have shown that both genetic and chemical strategies to block apoptotic signals can increase cell culture productivity. Here, we discuss various strategies which have been implemented to improve cellular viabilities and productivities in batch cultures.     

Alterations in cell nuclei during apoptosis

Apoptosis is a genetically programmed phenomenon that aids in maintaining homeostasis in multicellular organisms. The characteristic morphological features of apoptosis are highly conservative and are dependent on the cell type and the apoptotic inducer. The nuclear events occurring during apoptosis include changes at the molecular level (i.e. DNA cleavage, modifications of nuclear polypeptides, and proteolysis of several proteins important for cell maintenance), and, consequently, alterations at the morphological level (i.e. chromatin condensation, nuclear shrinkage, DNA fragmentation and apoptotic body formation). These events are still not fully understood. It is very probable that a progressive decrease in pH could also be an essential factor for the induction of nuclease and protease activities, and an important element of the optimal conditions for their function. This review details the current state of knowledge on apoptotic nuclear events, with particular focus on the proteins involved in the execution of apoptosis in cell nuclei, and on the differences in substrate cleavage profiles for different types of cell undergoing cell death.     

神经酰胺与细胞凋亡

在多细胞有机体中,凋亡对正常组织的发育和稳态是必需的。神经酰胺（ceramide,Cer）作为一种脂类分子,不仅是细胞膜的重要组成部分,而且是重要的凋亡参与者。在细胞膜上,它能够自发地聚集,形成招募死亡受体和配体的结构域从而促进信号的传导和放大;在细胞质中,它作为信号通路的中介者,通过许多不同的分子机制去调控机体的凋亡通路;在细胞核膜和核内,通过其代谢变化,决定细胞的增殖或凋亡;而且它的浓度和位置以及与其它分子的比例及其相互作用都会影响到细胞和组织的命运。综述了近来关于Cer及其代谢物的凋亡作用。     

Beta1 integrin-mediated T cell adhesion and cell spreading are regulated by calpain

To investigate the function of calpain in T cells, we sought to determine the role of this protease in cellular events mediated by beta1 integrins. T cell receptor cross-linked or phorbol ester-stimulated T cells binding to immobilized fibronectin induce the translocation of calpain to the cytoskeletal/membrane fraction of these cells. Such translocation of calpain is associated with proteolytic modification of protein tyrosine phosphatase 1B, increased cellular adhesion, and dramatic alterations in cellular morphology. However, affinity-related increases in T cell adhesion induced by the anti-beta1 integrin antibody 8A2 occur in a calpain-independent manner and in the absence of morphological shape changes. Furthermore, calpain undergoes activation in response to either alpha4beta1 or alpha5beta1 integrin binding to fibronectin in appropriately stimulated T cells, and calpain II as well as protein tyrosine phosphatase 1B accumulates at sites of focal contact formation. Inhibition of calpain activity not only inhibits the proteolytic modification of protein tyrosine phosphatase 1B, but also decreases the ability of T cells to adhere to and spread on immobilized fibronectin. Thus, we describe a potential regulatory role for calpain in beta1 integrin-mediated signaling events associated with T cell adhesion and cell spreading on fibronectin.     

Retinal endothelial cell apoptosis

Retinal endothelial cell (REC) apoptosis occurs in response to a number of stressors, including high glucose, oxidative stress, hypoxia. Because these stressors are common factors in a number of ocular diseases, it is critical to understand the cellular mechanisms by which apoptosis occurs in REC. This review discusses the various models of REC used in ophthalmological research. The mechanisms responsible for REC apoptosis are discussed, as well as potential therapeutics currently under development to prevent REC apoptosis. The primary goal of this review is provide the reader with a background knowledge of the current state of research ongoing in REC apoptosis and potential avenues for future testing.     

Re-expression of igf-ii is important for Beta cell regeneration in adult mice

Background

The key factors which support re-expansion of beta cell numbers after injury are largely unknown. Insulin-like growth factor II (IGF-II) plays a critical role in supporting cell division and differentiation during ontogeny but its role in the adult is not known. In this study we investigated the effect of IGF-II on beta cell regeneration.

Methodology/Principal Findings

We employed an in vivo model of ‘switchable’ c-Myc-induced beta cell ablation, pIns-c-MycER^TAM, in which 90% of beta cells are lost following 11 days of c-Myc (Myc) activation in vivo. Importantly, such ablation is normally followed by beta cell regeneration once Myc is deactivated, enabling functional studies of beta cell regeneration in vivo. IGF-II was shown to be re-expressed in the adult pancreas of pIns-c-MycER^TAM/IGF-II^+/+ (MIG) mice, following beta cell injury. As expected in the presence of IGF-II beta cell mass and numbers recover rapidly after ablation. In contrast, in pIns-c-MycER^TAM/IGF-II^+/− (MIGKO) mice, which express no IGF-II, recovery of beta cell mass and numbers were delayed and impaired. Despite failure of beta cell number increase, MIGKO mice recovered from hyperglycaemia, although this was delayed.

Conclusions/Significance

Our results demonstrate that beta cell regeneration in adult mice depends on re-expression of IGF-II, and supports the utility of using such ablation-recovery models for identifying other potential factors critical for underpinning successful beta cell regeneration in vivo. The potential therapeutic benefits of manipulating the IGF-II signaling systems merit further exploration.     

Growth and productivity of Beta vulgaris cell culture in fluidized bed reactors

Beta vulgaris cells were cultured in specially constructed fluidized bed reactors on Murashige and Skoog medium with 3% sucrose as a carbon source. Biomass density levels of approximately 17–18 g dry cell weight liter^–1 for 5 liter fermenters and 9–10 g dry cell weight liter^–1 for 50 liter reactor were observed. Depending on particular cell line, cell content of betalains reached levels ranging from 12 to 25 mg g dry cell weight^–1, representing the productivities of 14 to 17 mg of pigments per liter and day.The authors would like to thank the Chemap Tetra Laval group and the Kommission zur Förderung der wissenschaftlichen Forschung for the financial support of this project (KWF No. 2057.1).     

Mitochondria mediated cell death in diabetes

Mitochondrial dysfunction plays a role in the pathogenesis of a wide range of diseases that involve disordered cellular fuel metabolism and survival/death pathways, including neurodegenerative diseases, cancer and diabetes. Cytokine, virus recognition and cellular stress pathways converging on mitochondria cause apoptotic and/or necrotic cell death of β-cells in type-1 diabetes. Moreover, since mitochondria generate crucial metabolic signals for glucose stimulated insulin secretion (GSIS), mitochondrial dysfunction underlies both the functional derangement of GSIS and (over-nutrition) stress-induced apoptotic/necrotic β-cell death, hallmarks of type-2 diabetes. The apparently distinct mechanisms governing β-cell life/death decisions during the development of diabetes provide a remarkable example where remote metabolic, immune and stress signalling meet with mitochondria mediated apoptotic/necrotic death pathways to determine the fate of the β-cell. We summarize the main findings supporting such a pivotal role of mitochondria in β-cell death in the context of current trends in diabetes research.     

Beta4 integrin is required for hemidesmosome formation, cell adhesion and cell survival

The integrin heterodimer alpha 6 beta 4 is expressed in many epithelia and in Schwann cells. In stratified epithelia, alpha 6 beta 4 couple with BPAG1-e and BPAG2 to form hemidesmosomes, attaching externally to laminin and internally to the keratin cytoskeleton. To explore the function of this atypical integrin, and its relation to conventional actin-associated integrins, we targeted the removal of the beta 4 gene in mice. Tissues that express alpha 6 beta 4 are grossly affected. Stratified tissues are devoid of hemidesmosomes, display only a very fragile attachment to the basal lamina, and exhibit signs of degeneration and tissue disorganization. Simple epithelia which express alpha 6 beta 4 are also defective in adherence, even though they do not form hemidesmosomes. In the absence of beta 4, alpha 6 is dramatically downregulated, and other integrins do not appear to compensate for the loss of this heterodimer. These data have important implications for understanding integrin function in cell-substratum adhesion, cell survival and differentiation, and for understanding the role of alpha 6 beta 4 in junctional epidermolysis bullosa, an often lethal human disorder with pathology similar to our mice.