Concurrent isolation of granulocytes and lymphocytes with unaltered permeability, energy state, and metabolic capacity in vitro

A method has been developed based on sedimentation, discontinuous density gradient centrifugation, and differential lysis for the concurrent isolation from the same sample of blood of granulocytes and lymphocytes with yields of 27 and 38%, respectively. The isolated cellular fractions were characterized by a lack of cross-contamination and by the virtual absence of erythrocytes and platelets. Plasma membrane integrity, energy status, and metabolic capacity of the isolated cells were assessed by the cell contents of potassium, sodium, ATP, and by the uptake of oxygen. The respective values for these parameters of cell viability compared favorably with previously reported data for separately isolated granulocytes and lymphocytes. The cellular characteristics determined immediately after the isolation of the cells were maintained throughout a 2-hr period at room temperature.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/23272/1/0000209.pd

A local glucose-and oxygen concentration-based insulin secretion model for pancreatic islets

<p>Abstract</p> <p>Background</p> <p>Because insulin is the main regulator of glucose homeostasis, quantitative models describing the dynamics of glucose-induced insulin secretion are of obvious interest. Here, a computational model is introduced that focuses not on organism-level concentrations, but on the quantitative modeling of local, cellular-level glucose-insulin dynamics by incorporating the detailed spatial distribution of the concentrations of interest within isolated avascular pancreatic islets.</p> <p>Methods</p> <p>All nutrient consumption and hormone release rates were assumed to follow Hill-type sigmoid dependences on local concentrations. Insulin secretion rates depend on both the glucose concentration and its time-gradient, resulting in second-and first-phase responses, respectively. Since hypoxia may also be an important limiting factor in avascular islets, oxygen and cell viability considerations were also built in by incorporating and extending our previous islet cell oxygen consumption model. A finite element method (FEM) framework is used to combine reactive rates with mass transport by convection and diffusion as well as fluid-mechanics.</p> <p>Results</p> <p>The model was calibrated using experimental results from dynamic glucose-stimulated insulin release (GSIR) perifusion studies with isolated islets. Further optimization is still needed, but calculated insulin responses to stepwise increments in the incoming glucose concentration are in good agreement with existing experimental insulin release data characterizing glucose and oxygen dependence. The model makes possible the detailed description of the intraislet spatial distributions of insulin, glucose, and oxygen levels. In agreement with recent observations, modeling also suggests that smaller islets perform better when transplanted and/or encapsulated.</p> <p>Conclusions</p> <p>An insulin secretion model was implemented by coupling local consumption and release rates to calculations of the spatial distributions of all species of interest. The resulting glucose-insulin control system fits in the general framework of a sigmoid proportional-integral-derivative controller, a generalized PID controller, more suitable for biological systems, which are always nonlinear due to the maximum response being limited. Because of the general framework of the implementation, simulations can be carried out for arbitrary geometries including cultured, perifused, transplanted, and encapsulated islets.</p

Induction of Glucose Metabolism in Stimulated T Lymphocytes Is Regulated by Mitogen-Activated Protein Kinase Signaling

T lymphocytes play a critical role in cell-mediated immune responses. During activation, extracellular and intracellular signals alter T cell metabolism in order to meet the energetic and biosynthetic needs of a proliferating, active cell, but control of these phenomena is not well defined. Previous studies have demonstrated that signaling from the costimulatory receptor CD28 enhances glucose utilization via the phosphatidylinositol-3-kinase (PI3K) pathway. However, since CD28 ligation alone does not induce glucose metabolism in resting T cells, contributions from T cell receptor-initiated signaling pathways must also be important. We therefore investigated the role of mitogen-activated protein kinase (MAPK) signaling in the regulation of mouse T cell glucose metabolism. T cell stimulation strongly induces glucose uptake and glycolysis, both of which are severely impaired by inhibition of extracellular signal-regulated kinase (ERK), whereas p38 inhibition had a much smaller effect. Activation also induced hexokinase activity and expression in T cells, and both were similarly dependent on ERK signaling. Thus, the ERK signaling pathway cooperates with PI3K to induce glucose utilization in activated T cells, with hexokinase serving as a potential point for coordinated regulation

Selective effects of secretagogues on insulin secretion: A mathematical model

A mathematical model of the secretion of insulin from pancreatic islets of Langerhans is proposed. Previously proposed mathematical models of insulin secretion have dealt solely with glucose-stimulated release, and not with the more complex patterns of secretion (such as "off-responses") in response to other secretagogues or combinations of secretagogues.We conclude that (1) an off-response is consistent with a compartmental model; and (2) the facilitating effect of a constant concentration of glucose on leucine-stimulated insulin secretion is consistent with a selective effect of glucose on a single compartment of the model.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/24981/1/0000408.pd

The ERK and JNK pathways in the regulation of metabolic reprogramming.

Most tumor cells reprogram their glucose metabolism as a result of mutations in oncogenes and tumor suppressors, leading to the constitutive activation of signaling pathways involved in cell growth. This metabolic reprogramming, known as aerobic glycolysis or the Warburg effect, allows tumor cells to sustain their fast proliferation and evade apoptosis. Interfering with oncogenic signaling pathways that regulate the Warburg effect in cancer cells has therefore become an attractive anticancer strategy. However, evidence for the occurrence of the Warburg effect in physiological processes has also been documented. As such, close consideration of which signaling pathways are beneficial targets and the effect of their inhibition on physiological processes are essential. The MAPK/ERK and MAPK/JNK pathways, crucial for normal cellular responses to extracellular stimuli, have recently emerged as key regulators of the Warburg effect during tumorigenesis and normal cellular functions. In this review, we summarize our current understanding of the roles of the ERK and JNK pathways in controlling the Warburg effect in cancer and discuss their implication in controlling this metabolic reprogramming in physiological processes and opportunities for targeting their downstream effectors for therapeutic purposes.Brunel Research Initiative & Enterprise Fund, Brunel University of London (to CB), Kay Kendall Leukemia Fund (KKL443) (to CB), 250 Great Minds Fellowship, University of Leeds (to SP), AMMF Cholangiocarcinoma Charity (to SP and PMC), and Bloodwise (17014) (to SP and CB)