Multiple forms of porcine pancreatic α-amylase

Porcine pancreatic α-amylase can be fractionated into two components by DEAE-cellulose chromatography and by disc electrophoresis. The basis for fractionation is tentatively ascribed to a charge difference. The two components displayed the same specific activity and their thermal and pH stability, as well as the variation of V_max and K_m with pH, were identical within experimental error. It is concluded that the multiple forms of the amylase are physically distinct, but structurally related, with a common active site.     

Electrophysiology of pancreatic β-cells in intact mouse islets of Langerhans

When exposed to intermediate glucose concentrations (6–16 mol/l), pancreatic β-cells in intact islets generate bursts of action potentials (superimposed on depolarised plateaux) separated by repolarised electrically silent intervals. First described more than 40 years ago, these oscillations have continued to intrigue β-cell electrophysiologists. To date, most studies of β-cell ion channels have been performed on isolated cells maintained in tissue culture (that do not burst). Here we will review the electrophysiological properties of β-cells in intact, freshly isolated, mouse pancreatic islets. We will consider the role of ATP-regulated K⁺-channels (K_ATP-channels), small-conductance Ca²⁺-activated K⁺-channels and voltage-gated Ca²⁺-channels in the generation of the bursts. Our data indicate that K_ATP-channels not only constitute the glucose-regulated resting conductance in the β-cell but also provide a variable K⁺-conductance that influence the duration of the bursts of action potentials and the silent intervals. We show that inactivation of the voltage-gated Ca²⁺-current is negligible at voltages corresponding to the plateau potential and consequently unlikely to play a major role in the termination of the burst. Finally, we propose a model for glucose-induced β-cell electrical activity based on observations made in intact pancreatic islets.     

Ultrastructural responses of pancreatic β cells to metabolic alkalosis

Summary The ultrastructural changes in pancreatic cells were studied following glucose-induced insulin secretion in vitro, at two different extracellular pH (7.4 and 7.8). The pancreata perfused at pH 7.4 exhibited a biphasic insulin response to glucose challenge together with signs of increased emiocytotic activity and numerous microtubules in the cells. Conversely, the pancreata perfused at pH 7.8 showed a significant decrease in insulin secretion, and their cells revealed scarce emiocytotic images and a marked increase of intracellular granulolysis. These results represent the ultrastructural correlate of the reduced insulin secretion produced by metabolic alkalosis in the perfused rat pancreas.The authors wish to thank Mrs. Elma P. de Gagliardino and Mrs. Susana Rivas for excellent technical assistance.This research was partially supported by funds from CONICET and CIC, Pcia de Bs.As. C.L. Gómez Dumm, O.R. Rebolledo and J.J. Gagliardino are members of Carrera del Investigador del CONICET (Argentina)     

Control of pancreatic β cell regeneration by glucose metabolism

Recent studies revealed a surprising regenerative capacity of insulin-producing β cells in mice, suggesting that regenerative therapy for human diabetes could in principle be achieved. Physiologic β cell regeneration under stressed conditions relies on accelerated proliferation of surviving β cells, but the factors that trigger and control this response remain unclear. Using islet transplantation experiments, we show that β cell mass is controlled systemically rather than by local factors such as tissue damage. Chronic changes in β cell glucose metabolism, rather than blood glucose levels per se, are the main positive regulator of basal and compensatory β cell proliferation in vivo. Intracellularly, genetic and pharmacologic manipulations reveal that glucose induces β cell replication via metabolism by glucokinase, the first step of glycolysis, followed by closure of K(ATP) channels and membrane depolarization. Our data provide a molecular mechanism for homeostatic control of β cell mass by metabolic demand.     

Activation of double-stranded RNA-dependent protein kinase inhibits proliferation of pancreatic β-cells

Double-stranded RNA-dependent protein kinase (PKR) is revealed to participate in the development of insulin resistance in peripheral tissues in type 2 diabetes (T2DM). Meanwhile, PKR is also characterized as a critical regulator of cell proliferation. To date, no study has focused on the impact of PKR on the proliferation of pancreatic β-cells. Here, we adopted insulinoma cell lines and mice islet β-cells to investigate: (1) the effects of glucolipotoxicity and pro-inflammatory cytokines on PKR activation; (2) the effects of PKR on proliferation of pancreatic β-cells and its underlying mechanisms; (3) the actions of PKR on pro-proliferative effects of IGF-I and its underlying pathway. Our results provided the first evidence that PKR can be activated by glucolipitoxicity and pro-inflammatory cytokines in pancreatic β-cells, and activated PKR significantly inhibited cell proliferation by arresting cell cycle at G1 phase. Reductions in cyclin D1 and D2 as well as increases in p27 and p53 were associated with the anti-proliferative effects of PKR, and proteasome-dependent degradation took part in the reduction of cyclin D1 and D2. Besides, PKR activation abrogated the pro-proliferative effects of IGF-I by activating JNK and disrupting IRS1/PI3K/Akt signaling pathway. These findings indicate that the anti-proliferative actions of PKR on pancreatic β-cells may contribute to the pathogenesis of T2DM.     

Supplementary cryoprotective effect of carboxylated ε-poly-l-lysine during vitrification of rat pancreatic islets

This study was designed to investigate whether cryosurvival of rat pancreatic islets can be improved by carboxylated ε-poly-l-lysine (CPLL). Islets isolated from Wistar × Brown-Norway F1 rats (101–200 μm in diameter) were cryopreserved in three vitrification solutions containing ethylene glycol (EG; 30%, v/v) and CPLL (0%, 10%, or 20%, v/v) by Cryotop^® protocol (10 islets per device). The post-warm survival rate of the islets vitrified in the presence of 20% CPLL (74%), assessed by FDA/PI double staining, was higher than those in 0% and 10% CPLL (65% and 66%, respectively). Decreased EG concentrations (10% and 20%) in the presence of 20% CPLL resulted in impaired post-warm islet survival rates (50% and 64%, respectively). Value of stimulus index (SI) for 20 mM/3 mM glucose-stimulated insulin secretion was 4.1 in islets vitrified-warmed in the presence of 30% EG and 20% CPLL, which was comparable with those in fresh control islets and vitrified islets in 30% EG alone (4.1 and 4.4, respectively). A large number of islets (50 islets per device) could be cryopreserved in the presence of 30% EG and 20% CPLL by using nylon mesh as the device, without considerable loss of post-warm survival (68%) and SI value (3.7). In conclusion, supplementation of antifreeze 20% CPLL was effective in improving the post-warm survival of isolated rat pancreatic islets when vitrification solution containing 30% EG was used.     

Analyzing electrical activities of pancreatic β cells using mathematical models

Bursts of repetitive action potentials are closely related to the regulation of glucose-induced insulin secretion in pancreatic β cells. Mathematical studies with simple β-cell models have established the central principle that the burst-interburst events are generated by the interaction between fast membrane excitation and slow cytosolic components. Recently, a number of detailed models have been developed to simulate more realistic β cell activity based on expanded findings on biophysical characteristics of cellular components. However, their complex structures hinder our intuitive understanding of the underlying mechanisms, and it is becoming more difficult to dissect the role of a specific component out of the complex network. We have recently developed a new detailed model by incorporating most of ion channels and transporters recorded experimentally (the Cha-Noma model), yet the model satisfies the charge conservation law and reversible responses to physiological stimuli. Here, we review the mechanisms underlying bursting activity by applying mathematical analysis tools to representative simple and detailed models. These analyses include time-based simulation, bifurcation analysis and lead potential analysis. In addition, we introduce a new steady-state I-V (ssI-V) curve analysis. We also discuss differences in electrical signals recorded from isolated single cells or from cells maintaining electrical connections within multi-cell preparations. Towards this end, we perform simulations with our detailed pancreatic β-cell model.     

The role of autophagy in pancreatic β-cell and diabetes

Pancreatic β-cells play a key role in glucose homeostasis in mammals. Although large-scale protein synthesis and degradation occur in pancreatic β-cells, the mechanism underlying dynamic protein turnover in β-cells remains largely unknown. We found low-level constitutive autophagy in β-cells of C57BL/6 mice fed a standard diet; however, autophagy was markedly upregulated in mice fed a high-fat diet. β-cells of diabetic db/db mice contained large numbers of autophagosomes, compared with non-diabetic db/misty controls. The functional importance of autophagy was analyzed using β-cell-specific Atg7 knockout mice. Autophagy-deficient mice showed degeneration of β-cells and impaired glucose tolerance with reduced insulin secretion. While a high-fat diet stimulated β-cell autophagy in control mice, it induced a profound deterioration of glucose intolerance in β-cell autophagy-deficient mutants, partly because of the lack of a compensatory increase in β-cell mass. These results suggest that the degradation of unnecessary cellular components by autophagy is essential for maintenance of the architecture and function of β-cells. Autophagy also serves as a crucial element of stress responses to protect β-cells under insulin resistant states. Impairment of autophagic machinery could thus predispose individuals to type 2 diabetes.     

Effects of α-difluoromethylornithine on pancreatic growth induced by caerulein

The role of ornithine decarboxylase and of polyamines was investigated on caerulein-induced pancreatic growth by the use of α-difluoromethylornithine (DFMO), an enzyme-activated irreversible inhibitor of ornithine decarboxylase. By itself, DFMO did not affect the pancreatic gland at all but when combined with caerulein, it reduced the increases in DNA synthesis and DNA content initiated by the cholecystokinin analog. The general hypertrophic action of caerulein was not affected by DFMO but specific increases in amylase and chymotrypsin concentrations were observed after 2 days of caerulein. The effect on amylase concentration was further increased after 4 days but that on chymotrypsin was reversed, showing a significant decrease. These data suggest that the polyamines might be involved in pancreatic growth that is stimulated by caerulein and that their action could be mainly oriented towards cellularity. The specific decreases obtained in DNA synthesis and content brought about by DFMO support this observation.     

Adenosine signaling promotes regeneration of pancreatic β cells in vivo

Diabetes can be controlled with insulin injections, but a curative approach that restores the number of insulin-producing β cells is still needed. Using a zebrafish model of diabetes, we screened ~7,000 small molecules to identify enhancers of β cell regeneration. The compounds we identified converge on the adenosine signaling pathway and include exogenous agonists and compounds that inhibit degradation of endogenously produced adenosine. The most potent enhancer of β cell regeneration was the adenosine agonist 5'-N-ethylcarboxamidoadenosine (NECA), which, acting through the adenosine receptor A2aa, increased β cell proliferation and accelerated restoration of normoglycemia in zebrafish. Despite markedly stimulating β cell proliferation during regeneration, NECA had only a modest effect during development. The proliferative and glucose-lowering effect of NECA was confirmed in diabetic mice, suggesting an evolutionarily conserved role for adenosine in β cell regeneration. With this whole-organism screen, we identified components of the adenosine pathway that could be therapeutically targeted for the treatment of diabetes.     

Mechanism for antioxidative effects of thiazolidinediones in pancreatic β-cells

Thiazolidinediones (TZDs) are synthetic ligands of peroxisome proliferator-activated receptor-γ (PPARγ), a member of the nuclear receptor superfamily. TZDs are known to increase insulin sensitivity and also to have an antioxidative effect. In this study, we tested whether TZDs protect pancreatic β-cells from oxidative stress, and we investigated the mechanism involved in this process. To generate oxidative stress in pancreatic β-cells (INS-1 and βTC3) or isolated islets, glucose oxidase was added to the media. The extracellular and intracellular reactive oxygen species (ROS) were measured to directly determine the antioxidant effect of TZDs. The phosphorylation of JNK/MAPK after oxidative stress was detected by Western blot analysis, and glucose-stimulated insulin secretion and cell viability were also measured. TZDs significantly reduced the ROS levels that were increased by glucose oxidase, and they effectively prevented β-cell dysfunction. The antioxidative effect of TZDs was abolished in the presence of a PPARγ antagonist, GW9662. Real-time PCR was used to investigate the expression levels of antioxidant genes. The expression of catalase, an antioxidant enzyme, was increased by TZDs in pancreatic β-cells, and the knockdown of catalase significantly inhibited the antioxidant effect of TZDs. These results suggest that TZDs effectively protect pancreatic β-cells from oxidative stress, and this effect is dependent largely on PPARγ. In addition, the expression of catalase is increased by TZDs, and catalase, at least in part, mediates the antioxidant effect of TZDs in pancreatic β-cells.     

mTORC1 signaling and regulation of pancreatic β-cell mass

The capacity of β cells to expand in response to insulin resistance is a critical factor in the development of type 2 diabetes. Proliferation of β cells is a major component for these adaptive responses in animal models. The extracellular signals responsible for β-cell expansion include growth factors, such as insulin, and nutrients, such as glucose and amino acids. AKT activation is one of the important components linking growth signals to the regulation of β-cell expansion. Downstream of AKT, tuberous sclerosis complex 1 and 2 (TSC1/2) and mechanistic target of rapamycin complex 1 (mTORC1) signaling have emerged as prime candidates in this process, because they integrate signals from growth factors and nutrients. Recent studies demonstrate the importance of mTORC1 signaling in β cells. This review will discuss recent advances in the understanding of how this pathway regulates β-cell mass and present data on the role of TSC1 in modulation of β-cell mass. Herein, we also demonstrate that deletion of Tsc1 in pancreatic β cells results in improved glucose tolerance, hyperinsulinemia and expansion of β-cell mass that persists with aging.