In type 1 diabetes (T1D), there is a specific destruction of the insulin secreting pancreatic  cell. Although the exact molecular mechanisms underlying  cell destruction are not known, sera from T1D patients have been shown to promote Ca 2؉ -induced apoptosis. We now demonstrate that apolipoprotein CIII (apoCIII) is increased in serum from T1D patients and that this serum factor both induces increased cytoplasmic free intracellular Ca 2؉ R esearch over the last 30 years has established that type 1 diabetes (T1D) is an autoimmune disease, but the mechanisms͞events that trigger the initiation and progression of the disease are still not identified. Genetic, immunological, and environmental factors are involved in the pathogenesis of T1D and most likely the events involved differ between different patients. Voltage-gated L-type Ca 2ϩ channels have an important physiological role in pancreatic  cell signal transduction (1). These channels constitute an essential link between transient changes in membrane potential and insulin release. Changes in cytoplasmic free intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) in the  cell are associated with the activation of a spectrum of intracellular signals and are strictly regulated because prolonged high [Ca 2ϩ ] i is harmful to the cells. Sera from newly diagnosed T1D patients have been shown to increase the activity of voltage-gated L-type Ca 2ϩ channels in  cells, resulting in increased [Ca 2ϩ ] i upon depolarization and  cell apoptosis, effects that can be prevented by Ca 2ϩ channel blockers (2). The key question has been what factor in T1D serum that is responsible for the changes in [Ca 2ϩ ] i . In the present study, we have revealed the identity of a key factor in T1D sera that increases [Ca 2ϩ ] i as well as promotes apoptosis and found it to correspond to apolipoprotein CIII (apoCIII). The fact that not all sera from T1D patients affected [Ca 2ϩ ] i indicates that T1D is not caused by a single factor like apoCIII, which is in agreement with clinical observations, suggesting that different factors can act in concert with the autoimmune abnormalities resulting in  cell destruction. MethodsMedia. The basal medium used both for isolation of cells and for experiments was a Hepes buffer (pH 7.4), containing: 125 mM NaCl, 5.9 mM KCl, 1.3 mM CaCl 2 , 1.2 mM MgCl 2 , and 25 mM Hepes. BSA was added to the medium at a concentration of 1 mg͞ml. For cell culture, RPMI medium 1640 was supplemented with 100 g͞ml streptomycin, 100 units of penicillin, and 10% FCS, normal human, or diabetic serum.Preparation of Cells. Adult mice from a local colony (3) were starved overnight. Pancreatic islets were isolated by a collagenase technique, and cell suspensions were prepared as described (4, 5). Cells were seeded onto glass coverslips and cultured at 37°C in a humidified atmosphere of 5% CO 2 in air.Preparation and Purification of Sera. Sera from T1D patients and control subjects were collected, identically sterile-processed, and stored frozen at Ϫ20°C until used. The sera we...
Transthyretin (TTR) is a transport protein for thyroxine and, in association with retinol-binding protein, for retinol, mainly existing as a tetramer in vivo. We now demonstrate that TTR tetramer has a positive role in pancreatic -cell stimulus-secretion coupling. TTR promoted glucose-induced increases in cytoplasmic free Ca 2؉ concentration ([Ca 2؉ ]i) and insulin release. This resulted from a direct effect on glucose-induced electrical activity and voltagegated Ca 2؉ channels. TTR also protected against -cell apoptosis. The concentration of TTR tetramer was decreased, whereas that of a monomeric form was increased in sera from patients with type 1 diabetes. The monomer was without effect on glucose-induced insulin release and apoptosis. Thus, TTR tetramer constitutes a component in normal -cell function. Conversion of TTR tetramer to monomer may be involved in the development of -cell failure͞ destruction in type 1 diabetes.type 1 diabetes ͉ Ca 2ϩ channels ͉ insulin release ͉ -cell signal transduction ͉ apoptosis T ransthyretin (TTR) is a protein that is synthesized in the liver, the choroid plexus of the brain, and the endocrine pancreas (1, 2). It is a transport protein for thyroxine and, in association with retinol-binding protein, for retinol. It has been reported that 1-2% of plasma TTR circulates bound to highdensity lipoprotein (HDL) and that the association to the HDL vesicle occurs through binding to apolipoprotein A1 (3). TTR has a complex equilibrium between different quaternary structures in serum (4),but exists mainly as a tetrameric protein of 14-kDa subunits (160-380 mg͞liter) with only a small amount of TTR monomer present in vivo in normal individuals (5, 6). Consequently, measurements of TTR in serum by conventional methods mainly reflect the tetrameric form. The TTR amyloidoses are human diseases in which misfolded TTR protein aggregates in different tissues. Several point mutations in TTR have been related to familial amyloidotic polyneuropathy (FAP) (7). The fact that TTR is also produced within the pancreatic islet made us interested in evaluating a possible role of this protein in -cell stimulus-secretion coupling. MethodsIdentification of TTR in Human Sera. Sera from type 1 diabetes (T1D) patients and control subjects were collected, identically sterile-processed, heat-inactivated by incubation at 56°C for 30 min, and stored frozen at Ϫ20°C until used. Changes in cytoplasmic free Ca 2ϩ concentration ([Ca 2ϩ ] i ) were tested in -cells when they were depolarized with 25 mM KCl. Those diabetic sera that induced a higher increase in [Ca 2ϩ ] i than sera from controls were centrifuged, and the supernatant was passed through a 0.45-mm sterile filter. Samples were loaded on SepPak C 18 preconditioned with 0.1% trifluoroacetic acid. After application, the sample proteins were eluted with 60% acetonitrile in 0.1% trifluoroacetic acid. This procedure was repeated twice. The two fractions were pooled, and the volume was reduced by lyophilization.The lyophilized material was submitted to...
Methods to measure cellular target engagement are increasingly being used in early drug discovery. The Cellular Thermal Shift Assay (CETSA) is one such method. CETSA can investigate target engagement by measuring changes in protein thermal stability upon compound binding within the intracellular environment. It can be performed in high-throughput, microplate-based formats to enable broader application to early drug discovery campaigns, though high-throughput forms of CETSA have only been reported for a limited number of targets. CETSA offers the advantage of investigating the target of interest in its physiological environment and native state, but it is not clear yet how well this technology correlates to more established and conventional cellular and biochemical approaches widely used in drug discovery. We report two novel high-throughput CETSA (CETSA HT) assays for B-Raf and PARP1, demonstrating the application of this technology to additional targets. By performing comparative analyses with other assays, we show that CETSA HT correlates well with other screening technologies and can be applied throughout various stages of hit identification and lead optimization. Our results support the use of CETSA HT as a broadly applicable and valuable methodology to help drive drug discovery campaigns to molecules that engage the intended target in cells.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2025 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.