The Anx7(+/-) knockout mutation alters electrical and secretory responses to Ca(2+)-mobilizing agents in pancreatic ß-cells.

Insulin secretion from the pancreatic ß-cell is controlled by changes in membrane potential and intracellular Ca(2+). The contribution of intracellular Ca(2+) stores to this process is poorly understood. We have previously shown that ß-cells of mice lacking one copy of the Annexin 7 gene (Anx7(+/-)) express reduced levels of IP(3) receptors and defects in IP(3)-dependent Ca(2+) signaling. To further elucidate the effect of the Anx7(+/-) mutation on signaling related to intracellular Ca(2+) stores in the ß-cell, we measured the effects of Ca(2+) mobilizing agents on electrical activity, intracellular Ca(2+) and insulin secretion in control and mutant ß-cells. We found that the muscarinic agonist carbachol and the ryanodine receptor agonists caffeine and 4-chloro-m-cresol had more potent depolarizing effects on Anx7(+/-) ß-cells compared to controls. Accordingly, glucose-induced insulin secretion was augmented to a greater extent by caffeine in mutant islets. Surprisingly, ryanodine receptor-mediated Ca(2+) mobilization was not affected by the Anx7(+/-) mutation, suggesting that the mechanism underlying the observed differences in electrical and secretory responsiveness does not involve intracellular Ca(2+) stores. Our results provide evidence that both IP3 receptors and ryanodine receptors play important roles in regulating ß-cell membrane potential and insulin secretion, and that the Anx7(+/-) mutation is associated with alterations in the signaling pathways related to these receptors.

Impact of biotin supplemented diet on mouse pancreatic islet ß-cell mass expansion and glucose induced electrical activity.

Biotin supplemented diet (BSD) is known to enhance ß-cell replication and insulin secretion in mice. Here, we first describe BSD impact on the islet ß-cell membrane potential (Vm) and glucose-induced electrical activity. BALB/c female mice (n ≥ 20) were fed for nine weeks after weaning with a control diet (CD) or a BSD (100X). In both groups, islet area was compared in pancreatic sections incubated with anti-insulin and anti-glucagon antibodies; Vm was recorded in micro dissected islet ß-cells during perfusion with saline solutions containing 2.8, 5.0, 7.5-, or 11.0 mM glucose. BSD increased the islet and ß-cell area compared with CD. In islet ß-cells of the BSD group, a larger ΔVm/Δ[glucose] was found at sub-stimulatory glucose concentrations and the threshold glucose concentration for generation of action potentials (APs) was increased by 1.23 mM. Moreover, at 11.0 mM glucose, a significant decrease was found in AP amplitude, frequency, ascending and descending slopes as well as in the calculated net charge influx and efflux of islet ß-cells from BSD compared to the CD group, without changes in slow Vm oscillation parameters. A pharmacological dose of biotin in mice increases islet insulin cell mass, shifts islet ß-cell intracellular electrical activity dose response curve toward higher glucose concentrations, very likely by increasing KATP conductance, and decreases voltage gated Ca2+ and K+ conductance at stimulatory glucose concentrations.

Endosomal abnormalities related to amyloid precursor protein in cholesterol treated cerebral cortex neuronal cells derived from trisomy 16 mice, an animal model of Down syndrome.

The CNh and CTb cell lines are derived from the cerebral cortex of normal and trisomy 16 mice, an animal model of human trisomy 21, Down syndrome (DS), and represent in vitro models to study cellular events associated with the human condition. Amyloid precursor protein (APP) plays an important role in the development of neuropathology associated with DS and cholesterol in the amyloidogenic processing of APP. There is also increasing evidence of alterations in the recycling pathway of the early endosome compartment in nervous tissue from DS. In the present study, we report endosomal abnormalities related to amyloid precursor protein in cholesterol-treated CTb cells. Colocalization studies revealed the presence of APP-derived products in early endosomal compartments in both cell lines. Using internalization and immunoprecipitation techniques, differential effects were observed between the normal and trisomic cell lines when treated with cholesterol. Internalization experiments showed that the CTb cell line accumulates internalized APP in intracellular compartments for longer periods of time when compared to the CNh cell line. Immunoprecipitation revealed a differential interaction between the trafficking-related protein Rab4 and APP in the neuronal cell lines CNh and CTb. The present study suggests a putative mechanism by which overexpressed APP accumulates in intracellular compartments related to the endosomal trafficking pathway in individuals with DS, and highlights the usefulness of the CTb cell line as a model to study altered APP metabolism related to this genetic condition.

Ion channel formation by Alzheimer's disease amyloid beta-peptide (Abeta40) in unilamellar liposomes is determined by anionic phospholipids.

Incorporation of Alzheimer's disease amyloid beta-proteins (AbetaPs) across natural and artificial bilayer membranes leads to the formation of cation-selective channels. To study the peptide-membrane interactions involved in channel formation, we used cation reporter dyes to measure AbetaP-induced influx of Na+, Ca2+, and K+ into liposomes formed from phosphatidylserine (PS), phosphatidylinositol (PI) and phosphatidylcholine (PC). We found that Abeta40, but not Abeta40-1 or Abeta28, caused a dose-dependent increase in the concentration of each cation in the lumen of liposomes formed from the acidic phospholipids PS and PI. The Abeta40-induced changes in cation concentration, which we attribute to ion entry through Abeta40 channels, were not observed when using liposomes formed from the neutral phospholipid PC. Using mixtures of phospholipids, the magnitude of the AbetaP40-induced ion entry increased with the acidic phospholipid content of the liposomes, with entry being observed with as little as 5% PS or PI. Thus, while negatively charged phospholipids are required for formation of cation-permeable channels by Abeta40, a small amount is sufficient to support the process. These results have implications for the mechanisms of AbetaP cytotoxicity, suggesting that even a small amount of externalized negative charge could render cells susceptible to the deleterious effects of unregulated ion influx through AbetaP channels.

Loperamide mobilizes intracellular Ca2+ stores in insulin-secreting HIT-T15 cells.

1 We have investigated the effects of loperamide on intracellular Ca(2+) stores and membrane K(+) channels in insulin-secreting hamster insulinoma (HIT-T15) cells. 2 In cell-attached patch-clamp mode, loperamide (3-250 micro M) activated large single-channel currents. The loperamide-activated currents were tentatively identified as Ca(2+)-activated K(+) channel (K(Ca)) currents based on their single-channel conductance (145 pS), apparent reversal potential, and insensitivity to tolbutamide. Smaller single-channel currents with a conductance (32 pS) indicative of adenosine triphosphate-sensitive K(+) channels (K(ATP) channels) were also recorded, but were insensitive to loperamide. 3 Surprisingly, the loperamide-activated currents persisted in the absence of extracellular Ca(2+). Yet under these conditions, we still measured loperamide-induced Ca(2+) increases. These effects are dose dependent. Loperamide had no effects in the inside-out patch configuration, suggesting that loperamide does not directly activate the channels with large conductance, but does so secondarily to release of Ca(2+) from intracellular stores. 4 Carbachol (100 micro M), an agonist of muscarinic receptors, which mediates IP(3)-dependent intracellular Ca(2+) release, enhanced the effects of loperamide on K(Ca) channels. 5 Both the putative K(Ca) currents and Ca(2+) signals induced by loperamide (with '0' [Ca(2+)](o)) were abolished when the intracellular Ca(2+) stores had been emptied by pretreating the cells with either carbachol or thapsigargin, an endoplasmic reticulum Ca(2+)-ATPase inhibitor that blocks reuptake of calcium. 6 These data indicate that loperamide in insulin-secreting beta-cells evokes intracellular Ca(2+) release from IP(3)-gated stores and activates membrane currents that appear to be carried by K(Ca), rather than K(ATP) channels.

Pregnancy hormones prevent diabetes and reduce lymphocytic infiltration of islets in the NOD mouse.

Pregnancy is associated with a depression of the immune inflammatory system, and with increased growth and function of the pancreatic islets of Langerhans. We monitored glucosuria, blood glucose concentration, and lymphocytic infiltration of pancreatic islets in 30 female, 10-wk-old, pre-diabetic nonobese diabetic (NOD) mice divided into 3 treatment groups for 13 wk: group 1, saline; group 2, pregnancy hormones (dexamethasone 4 mg/Kg/day, progesterone 1.7 mg/Kg/day, growth hormone 0.6 mg/Kg/day, prolactin 1 mg/Kg/day, and estradiol 0.05 mg/Kg); and group 3, prolactin alone (1 mg/Kg/day). At sacrifice, the pancreases were fixed in paraformaldehyde and islet infiltration was evaluated. In the saline-treated group (#1) 4/10 mice developed diabetes, while in the hormone treated group (#2) none of the mice developed diabetes. Only 1/10 mice in the prolactin-treated group (#3) developed diabetes during the study. Islets from the hormone cocktail treated group were significantly less infiltrated than islets from the other 2 treatment groups (p <0.001). Thus, the pregnancy hormones protected NOD mice from developing diabetes and significantly reduced or eliminated insulitis and islet infiltration. Prolactin alone had a partial protective effect. The results have implications for prevention of type 1 diabetes and for immune suppression in patients receiving islet cell transplantation.

Apoptosis is directly related to intracellular amyloid accumulation in a cell line derived from the cerebral cortex of a trisomy 16 mouse, an animal model of Down syndrome.

Human Down syndrome (DS) represents the most frequent cause of mental retardation associated to a genetic condition. DS also exhibits a characteristic early onset of neuropathology indistinguishable from that observed in Alzheimer's disease (AD), namely the deposition of the beta-amyloid peptide. Early endosomal dysfunction has been described in individuals with DS and AD, suggesting an important role of this subcellular compartment in the onset and progression of the pathology. On the other hand, cholesterol activates the amyloidogenic processing pathway for the amyloid precursor protein, and the lipoprotein receptor-related peptide interacts with the beta-amyloid peptide. In the present work, using cell lines derived from the cortex of both normal and trisomy 16 mice (Ts16), an animal model of DS, we showed that the application of exogenous beta-amyloid has cytotoxic effects, expressed in decreased viability and increased apoptosis. Supplementation of the culture media with cholesterol associated to lipoprotein increased cell viability in both cell lines, but apoptosis decreased only in the normal cell line. Further, intracellular beta-amyloid content was elevated in trisomic cells following cholesterol treatment, with higher values in the trisomic cell line. Immunocytochemical detection showed intracellular accumulation of exogenous beta-amyloid in Rab4-positive compartments, which are known to be associated to endosomal recycling. The results suggest that the intracellular beta-amyloid pool plays a central role in apoptosis-mediated cell death in the trisomic condition.

Líquido cefalorraquídeo como medio de cultivo para xenotrasplantes / Cerebrospinal fluid as a culture medium for tissue transplantation

Se presenta la experiencia del trasplante de Islotes de Langerhans de ratón a un sistema de derivación ventriculoperitoneal previamente instalado en otra especie (llama). Se plantea que el Líquido cefalorraquídeo (LCR) es un ambiente privilegiado desde el punto de vista inmunitario, en el sentido de carecer de tasas elevadas de anticuerpos y células inmunoactivas. El objeto del trabajo es demostrar la variabilidad a largo plazo y la funcionalidad de estos islotes dentro del ambiente de LCR del receptor. Se observó producción de insulina durante todo el tiempo de observación sin existir indicios de rechazo inmunológico. Se discuten las implicancias y posible desarrollo posterior de este tipo de experiencias

Variaçöes da glicemia em crianças com diabetes insulino-dependente / Blood glucose variations in children with diabetes insulin-dependent

Nos Estados Unidos, no período do verao, acampamentos para crianças diabéticas sao patrocinados pela Associaçao Americana de Diabetes, como forma de promover o auto-controle da doença. Uma vez que o padrao de atividade da criança fica bastante alterado durante este período, a freqüência de hipoglicemias tende a ser um importante problema nestes acampamentos. Em 1990 realizou-se um levantamento no Glyndon Camp, Maryland, Estados Unidos, entre 282 crianças de 6 a 16 anos, com o objetivo de estabelecer os possíveis fatores associados a hipoglicemias (glicemias menores de 70 mg/dl, acompanhadas ou nao de sintomatologia). Essas crianças mediam suas glicemias, rotineiramente, pela manha em jejum, antes do almoço e do jantar e antes de dormir. Adicionalmente, media-se a glicemia quando a criança apresentava sintomatologia compatível com hipo ou hiperglicemia. Os resultados obtidos mostraram uma grande variaçao dos valores glicêmicos médios e do número de episódios de hipoglicemia nos 11 dias de acampamento. Os maiores valores glicêmicos médios ocorreram nos dias 1, 4 e 6 e nos dias 2, 5, 7, 8 e ll as médias foram significantemente menores. Hipoglicemias ocorreram com maior freqüência no 5( e 11( dias. Os menores valores glicêmicos ocorreram na medida feita antes de dormir, cuja média foi de 154 ñ 95 mg/dl. Os valores médios de glicemia foram menores e a freqüência de episódios de hipoglicemia foram maiores entre as crianças mais velhas, entre os meninos comparados com as meninas, nos negros, nas crianças com maior índice de massa corporal e naquelas que nao tiveram reduçao da dosagem de insulina injetada, durante o período de acampamento. Tais valores tenderam ainda a ser menores naquelas com hemoglobina glicosilada dentro da normalidade (l65 ñ 43 versus 188 ñ 48 mg/dl; p=O,O8), embora a frequência de hipoglicemias nao diferisse entre as duas categorias de contrqle metabólico. Este trabalho sugere que maior idade, sexo masculino e maior índice de massa corporal poderiam ser fatores predisponentes para hipoglicemia em acampamentos para crianças diabéticas.