Single-channel permeability and glycerol affinity of human aquaglyceroporin AQP3

For its fundamental relevance, transport of water and glycerol across the erythrocyte membrane has long been investigated before and after the discovery of aquaporins (AQPs), the membrane proteins responsible for water and glycerol transport. AQP1 is abundantly expressed in the human erythrocyte for maintaining its hydrohomeostasis where AQP3 is also expressed (at a level ~30-folds lower than AQP1) facilitating glycerol transport. This research is focused on two of the remaining questions: How permeable is AQP3 to water? What is the glycerol-AQP3 affinity under near-physiological conditions? Through atomistic modelling and large-scale simulations, we found that AQP3 is two to three times more permeable to water than AQP1 and that the glycerol-AQP3 affinity is approximately 500/M. Using these computed values along with the data from the latest literature on AQP1 and on erythrocyte proteomics, we estimated the water and glycerol transport rates across the membrane of an entire erythrocyte. We used these rates to predict the time courses of erythrocyte swelling-shrinking in response to inward and outward osmotic gradients. Experimentally, we monitored the time course of human erythrocytes when subject to an osmotic or glycerol gradient with light scattering in a stopped-flow spectrometer. We observed close agreement between the experimentally measured and the computationally predicted time courses of erythrocytes, which corroborated our computational conclusions on the AQP3 water-permeability and the glycerol-AQP3 affinity.     

Diffusion of glycerol through Escherichia coli aquaglyceroporin GlpF

The glycerol uptake facilitator, GlpF, a major intrinsic protein found in Escherichia coli, selectively conducts water and glycerol across the inner membrane. The free energy landscape characterizing the assisted transport of glycerol by this homotetrameric aquaglyceroporin has been explored by means of equilibrium molecular dynamics over a timescale spanning 0.12 μs. To overcome the free energy barriers of the conduction pathway, an adaptive biasing force is applied to the glycerol molecule confined in each of the four channels. The results illuminate the critical role played by intramolecular relaxation on the diffusion properties of the permeant. These free energy calculations reveal that glycerol tumbles and isomerizes on a timescale comparable to that spanned by its adaptive-biasing-force-assisted conduction in GlpF. As a result, reorientation and conformational equilibrium of glycerol in GlpF constitute a bottleneck in the molecular simulations of the permeation event. A profile characterizing the position-dependent diffusion of the permeant has been determined, allowing reaction rate theory to be applied for investigating conduction kinetics based on the measured free energy landscape.     

Pancreatic beta-cells: From generation to regeneration

The pancreas is composed of two main compartments consisting of endocrine and exocrine tissues. The majority of the organ is exocrine and responsible for the synthesis of digestive enzymes and for their transport via an intricate ductal system into the duodenum. The endocrine tissue represents less than 2% of the organ and is organized into functional units called islets of Langerhans, comprising alpha-, beta-, delta-, epsilon- and PP-cells, producing the hormones glucagon, insulin, somatostatin, ghrelin and pancreatic polypeptide (PP), respectively. Insulin-producing beta-cells play a central role in the control of the glucose homeostasis. Accordingly, absolute or relative deficiency in beta-cells may ultimately lead to type 1 and/or type 2 diabetes, respectively. One major goal of diabetes research is therefore to understand the molecular mechanisms controlling the development of beta-cells during pancreas morphogenesis, but also those underlying the regeneration of adult injured pancreas, and assess their significance for future cell-based therapy. In this review, we will therefore present new insights into beta-cell development with focus on beta-cell regeneration.     

Pancreatic islet beta-cells transiently metabolize pyruvate

Pancreatic beta-cell metabolism was followed during glucose and pyruvate stimulation of pancreatic islets using quantitative two-photon NAD(P)H imaging. The observed redox changes, spatially separated between the cytoplasm and mitochondria, were compared with whole islet insulin secretion. As expected, both NAD(P)H and insulin secretion showed sustained increases in response to glucose stimulation. In contrast, pyruvate caused a much lower NAD(P)H response and did not generate insulin secretion. Low pyruvate concentrations decreased cytoplasmic NAD(P)H without affecting mitochondrial NAD(P)H, whereas higher concentrations increased cytoplasmic and mitochondrial levels. However, the pyruvate-stimulated mitochondrial increase was transient and equilibrated to near-base-line levels. Inhibitors of the mitochondrial pyruvate-transporter and malate-aspartate shuttle were utilized to resolve the glucose- and pyruvate-stimulated NAD(P)H response mechanisms. These data showed that glucose-stimulated mitochondrial NAD(P)H and insulin secretion are independent of pyruvate transport but dependent on NAD(P)H shuttling. In contrast, the pyruvate-stimulated cytoplasmic NAD(P)H response was enhanced by both inhibitors. Surprisingly the malate-aspartate shuttle inhibitor enabled pyruvate-stimulated insulin secretion. These data support a model in which glycolysis plays a dominant role in glucose-stimulated insulin secretion. Based on these data, we propose a mechanism for glucose-stimulated insulin secretion that includes allosteric inhibition of tricarboxylic acid cycle enzymes and pH dependence of mitochondrial pyruvate transport.     

Free-energy landscape of glycerol permeation through aquaglyceroporin GlpF determined from steered molecular dynamics simulations

The free-energy landscape of glycerol permeation through the aquaglyceroporin GlpF has been estimated in the literature by the nonequilibrium method of steered molecular dynamics (SMD) simulations and by the equilibrium method of adaptive biasing force (ABF) simulations. However, the ABF results qualitatively disagree with the SMD results that were based on the Jarzynski equality (JE) relating the equilibrium free-energy difference to the nonequilibrium work of the irreversible pulling experiments. In this paper, I present a new SMD study of the glycerol permeation through GlpF to explore the free-energy profile of glycerol along the permeation channel. Instead of the JE in terms of thermodynamic work, I use the fluctuation-dissipation theorem (FDT) of Brownian dynamics (BD), in terms of mechanical work, for extracting the free-energy difference from the nonequilibrium work of irreversible pulling experiments. The results of this new SMD-BD-FDT study are in agreement with the experimental data and with the ABF results.     

The Arabidopsis thaliana aquaglyceroporin AtNIP7;1 is a pathway for arsenite uptake

We studied the effect of loss of function in the NIP subfamily II in Arabidopsis thaliana to assess their potential role(s) in arsenite (AsIII) uptake. Loss of function in AtNIP7;1 led to increased plant tolerance to AsIII and reduced total As in planta. AtNIP7;1 expression in various yeast backgrounds increased AsIII sensitivity. In the acr3Δ yeast genotype, AtNIP7;1 caused a moderate increase in AsV tolerance. Short-term As uptake in fsp1Δ expressing AtNIP7;1 was significantly larger than that in the empty vector control.

The data suggest that AtNIP7;1 can mediate AsIII transport and contributes to AsIII uptake in plants.     

Acyrthosiphon pisum AQP2: a multifunctional insect aquaglyceroporin

Annotation of the recently sequenced genome of the pea aphid (Acyrthosiphon pisum) identified a gene ApAQP2 (ACYPI009194, Gene ID: 100168499) with homology to the Major Intrinsic Protein/aquaporin superfamily of membrane channel proteins. Phylogenetic analysis suggests that ApAQP2 is a member of an insect-specific clade of this superfamily. Homology model structures of ApAQP2 showed a novel array of amino acids comprising the substrate selectivity-determining "aromatic/arginine" region of the putative transport pore. Subsequent characterization of the transport properties of ApAQP2 upon expression in Xenopus oocytes supports an unusual substrate selectivity profile. Water permeability analyses show that the ApAQP2 protein exhibits a robust mercury-insensitive aquaporin activity. However unlike the water-specific ApAQP1 protein, ApAQP2 forms a multifunctional transport channel that shows a wide permeability profile to a range of linear polyols, including the potentially biologically relevant substrates glycerol, mannitol and sorbitol. Gene expression analysis indicates that ApAQP2 is highly expressed in the insect bacteriocytes (cells bearing the symbiotic bacteria Buchnera) and the fat body. Overall the results demonstrate that ApAQP2 is a novel insect aquaglyceroporin which may be involved in water and polyol transport in support of the Buchnera symbiosis and aphid osmoregulation.     

Role of single-channel stochastic noise on bursting clusters of pancreatic beta-cells.

To study why pancreatic beta-cells prefer to burst as a multi-cellular complex, we have formulated a stochastic model for bursting clusters of excitable cells. Our model incorporated a delayed rectifier K+ channel, a fast voltage-gated Ca2+ channel, and a slow Cai-blockable Ca2+ channel. The fraction of ATP-sensitive K+ channels that may still be active in the bursting regime was included in the model as a leak current. We then developed an efficient method for simulating an ionic current component of an excitable cell that contains several thousands of channels opening simultaneously under unclamped voltage. Single channel open-close stochastic events were incorporated into the model by use of binomially distributed random numbers. Our simulations revealed that in an isolated beta-cell [Ca2+]i oscillates with a small amplitude about a low [Ca2+]i. However, in a large cluster of tightly coupled cells, stable bursts develop, and [Ca2+]i oscillates with a larger amplitude about a higher [Ca2+]i. This may explain why single beta-cells do not burst and also do not release insulin.     

Implications of aquaglyceroporins 7 and 9 in glycerol metabolism and metabolic syndrome

The discovery of water channel protein (aquaporin [AQP]) has made a great impact on life sciences. So far, 13 AQPs have been identified in human. AQP3, 7, 9, and 10 are subcategorized as aquaglyceroporins which permeabilize glycerol as well as water. Many investigators have demonstrated that AQPs play a crucial role in the maintenance of water homeostasis, but the physiological significance of some AQPs as glycerol channels remains elusive. Adipocyte is a major source of glycerol, which is one of the substrates for hepatic gluconeogenesis. This review focuses on recent studies on glycerol metabolism through AQP7 and AQP9, and briefly discusses the importance of glycerol channel in adipocytes, liver, and heart.     

The Role of Exosomes in Pancreatic Cancer Microenvironment

Exosomes are nanovesicles shed by cells as a means of communication with other cells. Exosomes contain mRNAs, microRNAs (miRs) and functional proteins. In the present paper, we develop a mathematical model of tumor–immune interaction by means of exosomes shed by pancreatic cancer cells and dendritic cells. Cancer cells’ exosomes contain miRs that promote their proliferation and that inhibit immune response by dendritic cells, and by CD4+ and CD8+ T cells. Dendritic cells release exosomes with proteins that induce apoptosis of cancer cells and that block regulatory T cells. Simulations of the model show how the size of the pancreatic cancer can be determined by measurement of specific miRs (miR-21 and miR-203 in the case of pancreatic cancer), suggesting these miRs as biomarkers for cancer.     

The Role of Fibroblasts in Pancreatic Cancer: Extracellular Matrix Versus Paracrine Factors

BACKGROUND AND AIM: Desmoplasia is a characteristic feature and a suspected mechanism of tumor progression in pancreatic ductal adenocarcinoma (PDAC). Main constituents of the stroma involve cancer-associated fibroblasts (CAFs) and extracellular matrix (ECM). The aim of this study was to dissect the interaction of CAFs, ECM, and PDAC cells in both an in vitro setting and a large-scale clinical cohort study. METHODS AND MATERIAL: Patients operated for PDAC were identified from our prospectively maintained clinical database. A standard pathology protocol was applied for pancreatoduodenectomy specimens also assessing CAF activation as either CAF grade 0 or CAF grade +. Interaction between a spectrum of pancreatic cancer cell lines (PCCs) and mouse embryonic fibroblasts (NIH 3T3) was assessed in a conditioned medium experimental setup. RESULTS: One hundred eleven patients operated for PDAC from 2001 to 2011 were identified. Univariate analysis disclosed CAF grade + (P = .030), positive M status (P < .001), and lymph node ratio (LNR) > 0.1 (P = .045) to impair overall survival. Independent prognostic factors were CAF grade (P = .050) and positive M status (P = .002). CAF grade correlated with N status (CC = 0.206, P = .030), LNR (CC = 0.187, P = .049), tumor size (CC = ?0.275, P = .003), and M status (CC = 0.190, P = .045). In the in vitro setting, paracrine effects of pancreatic cancer cell resulted in morphological activation of fibroblasts and tumor cell differentiation–dependent increase of fibroblast growth. Paracrine effects of poorly differentiated PCCs led to an upregulation of Vimentin in NIH 3T3 fibroblasts. Paracrine effects of fibroblasts on their part promoted cancer cell motility in all PCCs. As the second stromal component, fibroblast-derived ECM resulted in significantly decreased proliferation depending on density and led to upregulation of ZEB1 in poorly differentiated PCCs. CONCLUSION: In PDAC patients, positive CAF grading was identified as a negative prognostic parameter correlating with positive N status, high LNR, positive M status, and smaller tumor size. Whereas bilateral interaction of PCCs and CAFs promotes tumor progression, ECM poses PCC growth restrictions. In summary, our study discloses differential effects of stromal components and may help to interpret heterogeneous results of former studies.     

The Role of Gastrin and CCK Receptors in Pancreatic Cancer and other Malignancies

The gastrointestinal (GI) peptide gastrin is an important regulator of the release of gastric acid from the stomach parietal cells and it also plays an important role in growth of the gastrointestinal tract. It has become apparent that gastrin and its related peptide cholecystokinin (CCK) are also significantly involved with growth of GI cancers as well as other malignancies through activation of the cholecystokinin-B (CCK-B) receptor. Of interest, gastrin is expressed in the embryologic pancreas but not in the adult pancreas; however, gastrin becomes re-expressed in pancreatic cancer where it stimulates growth of this malignancy by an autocrine mechanism. Strategies to down-regulate gastrin or interfere with its interface with the CCK receptor with selective antibodies or receptor antagonists hold promise for the treatment of pancreatic cancer and other gastrin - responsive tumors.     

Folding and stability of the aquaglyceroporin GlpF: Implications for human aqua(glycero)porin diseases

Aquaporins are highly selective polytopic transmembrane channel proteins that facilitate the permeation of water across cellular membranes in a large diversity of organisms. Defects in aquaporin function are associated with common diseases, such as nephrogenic diabetes insipidus, congenital cataract and certain types of cancer. In general, aquaporins have a highly conserved structure; from prokaryotes to humans. The conserved structure, together with structural dynamics and the structural framework for substrate selectivity is discussed. The folding pathway of aquaporins has been a topic of several studies in recent years. These studies revealed that a conserved protein structure can be reached by following different folding pathways. Based on the available data, we suggest a complex folding pathway for aquaporins, starting from the insertion of individual helices up to the formation of the tetrameric aquaporin structure. The consequences of some known mutations in human aquaporin-encoding genes, which most likely affect the folding and stability of human aquaporins, are discussed.     

Dihydroxyacetone and methylglyoxal as permeants of the Plasmodium aquaglyceroporin inhibit parasite proliferation

The aquaglyceroporin of Plasmodium falciparum (PfAQP) is a bi-functional channel with permeability for water and solutes. Its functions supposedly are in osmotic protection of parasites and in facilitation of glycerol permeation for glycerolipid biosynthesis. Here, we show PfAQP permeability for the glycolysis-related metabolites methylglyoxal, a cytotoxic byproduct, and dihydroxyacetone, a ketotriose. AQP3, the red cell aquaglyceroporin, also passed dihydroxacetone but excluded methylglyoxal. Proliferation of malaria parasites was inhibited by methylglyoxal with an IC50 around 200 microM. Surprisingly, also dihydroxyacetone, which is an energy source in human cells, was antiproliferative in chloroquine-sensitive and resistant strains with an IC50 around 3 mM. We expressed P. falciparum glyceraldehyde 3-phosphate dehydrogenase (PfGAPDH) to examine whether it is inhibited by either carbonyl compound. Methylglyoxal did not affect PfGAPDH on incubation with 2.5 mM for 20 h. Treatment with 2.5 mM dihydroxyacetone, however, abolished PfGAPDH activity within 6 h. Aquaglyceroporin permeability for glycolytic metabolites may thus be of physiological significance.     

Drug uptake and modulation of drug resistance in Leishmania by an aquaglyceroporin

Leishmaniasis is a protozoan parasitic disease that affects 12 million people worldwide. The first line choice for the treatment of this disease is antimonial drugs. In the endemic regions, resistance to this class of drugs is a major impediment to treatment. Microbes often become resistant to drugs by mutation or down-regulation of uptake systems, but the uptake system for the antimonial drugs in Leishmania is unknown. In other organisms, aquaglyceroporins have been shown to facilitate uptake of trivalent metalloids. In this study, we report the identification and characterization of aquaglyceroporins from Leishmania major (LmAQP1) and Leishmania tarentolae (LtAQP1), respectively. These Leishmania proteins have the conserved signature motifs of aquaglyceroporins. Transfection of LmAQP1 into three species of Leishmania, L. tarentolae, Leishmania infantum, and L. major, produced hypersensitivity to both As(III) and Sb(III) in all three strains. Increased production of LmAQP1 was detected by immunoblotting. Drug-resistant parasites with various mutations leading to resistance mechanisms became hypersensitive to both metalloids after expression of LmAQP1. Increased rates of uptake of As(III) or Sb(III) correlated with metalloid sensitivity of the wild type and drug-resistant transfectants. Transfection of LmAQP1 in a Pentostam-resistant field isolate also sensitized the parasite in the macrophage-associated amastigote form. One allele of LmAQP1 was disrupted in L. major, and the resulting cells became 10-fold more resistant to Sb(III). This is the first report of the uptake of a metalloid drug by an aquaglyceroporin in Leishmania, suggesting a strategy to reverse resistance in the field.     

Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients ¹, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristic, organ procurement and preservation affect the isolation outcome ². At University of Illinois at Chicago (UIC) we have developed a successful isolation protocol with an improved purification gradient ³. The program started in January 2004, and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) ^4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al ⁸. Briefly, after cleaning the pancreas from the surrounding tissue, it was perfused with enzyme solution (Serva Collagenase + Neutral Protease or Sigma V enzyme). The distended pancreas was then transferred to the Ricordi digestion chamber, connected to a modified, closed circulation tubing system, and warmed up to 37°C. During the digestion, the chamber was shaken gently. Samples were taken continuously to monitor the digestion progress. Once free islets were detected under the microscope, the digestion was stopped by flushing cold (4°C) RPMI dilution solution (Mediatech, Herndon, VA) into the circulation system to dilute the enzyme. After being collected and washed in M199 media supplemented with human albumin, the tissue was sampled for pre-purification count and incubated with UW solution before purification. Purification process will be described in Part II: Purification and Culture of Human Islets.