Regulation of hepatic function by brain neuropeptides

ＲｅｇｕｌａｔｉｏｎｏｆｈｅｐａｔｉｃｆｕｎｃｔｉｏｎｂｙｂｒａｉｎｎｅｕｒｏｐｅｐｔｉｄｅｓＭａｓａｓｈｉＹｏｎｅｄａＳｕｂｊｅｃｔｈｅａｄｉｎｇｓｌｉｖｅｒ／ｐｈｙｓｉｏｌｏｇｙ；ｔｈｙｒｏｔｒｏｐｉｎｒｅｌｅａｓｉｎｇｈｏｒｍｏｎｅ；ｃｏｒｔｉ...     

Genetic modification of hypertension by sGCα1

Hypertension is an important modifiable risk factor for coronary heart disease, congestive heart failure, stroke, end-stage renal disease, and peripheral vascular disease, but many of the molecular mechanisms and genetic factors underlying the development of the most common forms of human hypertension remain to be defined. Abundant evidence suggests that nitric oxide (NO) and one of its primary targets, the cyclic guanosine monophosphate (cGMP)-generating enzyme soluble guanylate cyclase (sGC), have a critical role in regulating blood pressure. The availability of murine models of hypertension and the revolution in human genetics research (e.g., genome-wide association studies [GWAS]), resulting in the identification of dozens of genetic loci that affect normal variation in blood pressure and susceptibility to hypertension, provide a unique opportunity to dissect the mechanisms by which NO-cGMP signaling regulates blood pressure and to gain important insights into the pathogenesis of hypertension. In this review, we will give an overview of the current knowledge relating to the role of sGC in the regulation of blood pressure, discussing data obtained from genetically modified mouse models as well as from human genetic studies.     

Differentiation of Mycobacterium species by restriction enzyme analysis of amplified 16S–23S ribosomal DNA spacer sequences

Setting: Mycobacteriology research and service laboratories in Thailand.Objective: To evaluate the possibility of differentiating species of mycobacteria by amplifying 16S–23S ribosomal deoxyribonucleic acid (DNA) spacer and restriction enzyme analysis of the products.Design: DNA of 113 strains of mycobacteria belonging to 18 species of the genus Mycobacterium were amplified by primers PL1 (5′-GAAGTCGTAACAAGG) and PL2 (5′-CAAGGCATCCACCAT). The amplified products as well as their HaeIII-, MspI- and BstXI-digested products were visualized after agarose gel electrophoresis.Results: The amplified products of rapid-growing mycobacteria were different from the slow-growing mycobacteria. The restriction profiles of members of M. tuberculosis complex were the same as each other but different from other investigated species. The restriction profiles of some species, such as M. avium, M. intracellulare and M. gordonae, were unique, while those of the other species had more than one pattern. However, the restriction profiles of most investigated species were different from each other.Conclusion: This preliminary study suggested that the method might be useful for species differentiation of some commonly isolated pathogenic mycobacteria.     

Regulation of pancreatic β-cell survival by nitric oxide

The reduction of pancreatic β-cell mass is an important factor in the development of type 1 and type 2 diabetes. Understanding the mechanisms that regulate the maintenance of pancreatic β-cell mass as well as β-cell death is necessary for the establishment of therapeutic strategies. In this context, nitric oxide (NO) is a diatomic, gaseous, highly reactive molecule with biological activity that participates in the regulation of pancreatic β-cell mass. Two types of cellular responses can be distinguished depending on the level of NO production. First, pancreatic β-cells exposed to inflammatory cytokines, lipid stress or hyperglycaemia produce high concentrations of NO, mainly due to the activation of inducible NO synthase (iNOS), thus promoting cell death. Meanwhile, under homeostatic conditions, low concentrations of NO, constitutively produced by endothelial NO synthase (eNOS), promote cell survival. Here, we will discuss the current knowledge of the NO-dependent mechanisms activated during cellular responses, emphasizing those related to the regulation of cell survival.     

Regulation of calreticulin–major histocompatibility complex (MHC) class I interactions by ATP

The MHC class I peptide loading complex (PLC) facilitates the assembly of MHC class I molecules with peptides, but factors that regulate the stability and dynamics of the assembly complex are largely uncharacterized. Based on initial findings that ATP, in addition to MHC class I-specific peptide, is able to induce MHC class I dissociation from the PLC, we investigated the interaction of ATP with the chaperone calreticulin, an endoplasmic reticulum (ER) luminal, calcium-binding component of the PLC that is known to bind ATP. We combined computational and experimental measurements to identify residues within the globular domain of calreticulin, in proximity to the high-affinity calcium-binding site, that are important for high-affinity ATP binding and for ATPase activity. High-affinity calcium binding by calreticulin is required for optimal nucleotide binding, but both ATP and ADP destabilize enthalpy-driven high-affinity calcium binding to calreticulin. ATP also selectively destabilizes the interaction of calreticulin with cellular substrates, including MHC class I molecules. Calreticulin mutants that affect ATP or high-affinity calcium binding display prolonged associations with monoglucosylated forms of cellular MHC class I, delaying MHC class I dissociation from the PLC and their transit through the secretory pathway. These studies reveal central roles for ATP and calcium binding as regulators of calreticulin–substrate interactions and as key determinants of PLC dynamics.MHC class I molecules are ligands for the antigen receptors of CD8⁺ T cells and natural killer cells. The assembly and folding of MHC class I molecules with antigenic peptides take place within the endoplasmic reticulum (ER) and are facilitated by a multiprotein complex called the peptide loading complex (PLC) (reviewed in 1, 2). Structurally, the PLC involves the association of MHC class I heterodimers with the transporter associated with antigen processing (TAP), an interaction bridged, via several protein–protein interactions, within the ER lumen. Tapasin interacts with TAP via its transmembrane domain and with MHC class I via its ER luminal domains. MHC class I molecules also interact with the glycan-binding chaperone calreticulin through a conserved glycan on the α2-domain of the MHC class I heavy chain (3, 4). The thiol oxidoreductase ERp57, which functions as a cellular cochaperone for calreticulin (CRT), forms a disulfide-linked heterodimer with tapasin (reviewed in 1). In this manner, by bridging interactions with MHC class I and tapasin, respectively, calreticulin and ERp57 stabilize the binding of MHC class I molecules to tapasin–TAP complexes (reviewed in 1, 2). Cellular deficiencies in calreticulin and ERp57 destabilize MHC class I interactions with other components of the PLC (5–8). Furthermore, peptide binding to MHC class I has been shown to destabilize MHC class I–tapasin interactions (9, 10), but how other cellular factors influence PLC stability is largely uncharacterized.In this investigation, we show that ATP destabilizes MHC class I interactions with PLC components. ATP is a known regulator of substrate interactions with several cellular chaperones. Calreticulin, like several other chaperones, is known to interact with ATP (8, 11, 12). However, the location of the calreticulin-ATP–binding site is unknown, as is the influence of ATP on calreticulin binding to cellular substrates, including MHC class I. In this study, using computational methods validated by experimental approaches, we identify residues within the globular domain of calreticulin that are important for ATP binding and ATPase activity. Based on further investigations into the functional effects of calreticulin mutants with deficiencies in ATP interactions, we elucidate a key role for ATP in the regulation of PLC dynamics and the interaction of calreticulin with other cellular proteins.     

Emergent ultra–long-range interactions between active particles in hybrid active–inactive systems

Particle–particle interactions determine the state of a system. Control over the range of such interactions as well as their magnitude has been an active area of research for decades due to the fundamental challenges it poses in science and technology. Very recently, effective interactions between active particles have gathered much attention as they can lead to out-of-equilibrium cooperative states such as flocking. Inspired by nature, where active living cells coexist with lifeless objects and structures, here we study the effective interactions that appear in systems composed of active and passive mixtures of colloids. Our systems are 2D colloidal monolayers composed primarily of passive (inactive) colloids, and a very small fraction of active (spinning) ferromagnetic colloids. We find an emergent ultra–long-range attractive interaction induced by the activity of the spinning particles and mediated by the elasticity of the passive medium. Interestingly, the appearance of such interaction depends on the spinning protocol and has a minimum actuation timescale below which no attraction is observed. Overall, these results clearly show that, in the presence of elastic components, active particles can interact across very long distances without any chemical modification of the environment. Such a mechanism might potentially be important for some biological systems and can be harnessed for newer developments in synthetic active soft materials.Active-matter systems have received much interest due to their emergent nonequilibrium phase and collective dynamical behavior. This interest is well founded as some of the most ubiquitous and important biological systems or processes can exhibit such emergent nonequilibrium behavior, which is perhaps most recognizable in macroscopic examples ranging from schools of aquatic organisms like rays or fish (1–3), herds of livestock (4), flocks of birds (5–7), and even a mosh pit at a heavy metal concert (8). Active-matter systems are additionally unique in that such phenomena spans multiple length scales from meter to nanometer. At these smaller length scales, it is clear that such dynamical adaptation and phase separation are necessary to perform many vital biological processes. Dense crowds of cells move collectively through tissue during development and in many of the immune response processes, i.e., wound healing (9). Sea urchin sperm cells have been found to phase separate and organize into arrays of vortices when the density of spermatozoa is large enough (10). In fact, a myriad of biological systems, and experimental systems with biological components, have reported swarming (11–13), flocking (6, 14, 15), spiraling (13, 16), and many more nonequilibrium steady states (17, 18). It is clear that, in all these systems, a combination of the activity, shape of the active agents, and the environment lead to effective out-of-equilibrium interactions that determine their steady states. These active-matter systems have been particularly attractive for theoretical studies as there are relatively few artificial systems that can mimic these active-matter systems, in addition to a wide range of parameter space that can be explored. Simulations have been able to capture a wide range of collective dynamical behavior and spontaneous phase separation in these nonequilibrium systems (19–22).Although most experimental and simulation studies have focused on systems composed of purely active components (23), here we investigate a hybrid system composed of active and passive components. This hybrid active–passive system serves as an excellent model for collective motion in complex, crowded biological environments where active units must move through or past nonmotile cells or tissues. Such a model system could potentially help to explain collective motion and phase separation observed in bacterial biofilms (24) and cells migrating through tissue interfaces or within tissues and cell sheets (25–29). In the past, there have been very few studies investigating hybrid active–passive systems and almost all of them are simulation or theoretical studies. Interestingly, these studies have reported nonequilibrium phase segregation in systems of motile and nonmotile rods (21), passive spheres and active rods (22), active and passive agents (15, 29), and active and passive hard spheres (30). Attempts to observe similar behavior in experimental artificial model systems have proved to be a more difficult task, presumably due to the fact that other interactions are present in such systems compared with the relatively simple models that theory and simulation have considered. Despite this, there are many systems, both experimental and theoretical, that show an emergent attraction between active agents in mixtures of active and passive populations and subsequent phase separation. Nevertheless, the origin of this nonequilibrium phase segregation is not well understood. Here, we clearly show that two active spinning particles in a dense monolayer of passive colloids attract, and such attraction can be felt at extremely long distances. This range is much longer than the magnetic dipole–dipole interaction and tunable via the activity and kinetics of the system. The origin of such long-range attraction is due to the elasticity of the monolayer. Our results, thus, provide much-needed understanding of the emergent interactions in synthetic mixed active systems and can help distinguish what biological interactions can be due to purely physical phenomena and which interactions require presumably physical and biological/biochemical stimuli.     

Regulation of S100A10 by the PML-RAR-α oncoprotein

Acute promyelocytic leukemia (APL) is a distinct subtype of acute myeloid leukemia that results from the expression of the promyelocytic leukemia-retinoic acid receptor α (PML-RAR-α) oncoprotein. It is characterized by severe hemorrhagic complications due in part to excessive fibrinolysis, resulting from the excessive generation of the fibrinolytic enzyme, plasmin, at the cell surface of the PML cells. The treatment of patients with all-trans retinoic acid (ATRA) effectively ameliorates the disease by promoting the destruction of the PML-RAR-α oncoprotein. In the present study we show for the first time that the plasminogen receptor, S100A10, is present on the extracellular surface of APL cells and is rapidly down-regulated in response to all-trans retinoic acid. The loss of S100A10 is concomitant with a loss in fibrinolytic activity. Furthermore, the induced expression of the PML-RAR-α oncoprotein increased the expression of cell surface S100A10 and also caused a dramatic increase in fibrinolytic activity. Depletion of S100A10 by RNA interference effectively blocked the enhanced fibrinolytic activity observed after induction of the PML-RAR-α oncoprotein. These experiments show that S100A10 plays a crucial role in the generation of plasmin leading to fibrinolysis, thus providing a link to the clinical hemorrhagic phenotype of APL.     

Regulation of oxidative DNA damage repair by DNA polymerase λ and MutYH by cross-talk of phosphorylation and ubiquitination

It is of pivotal importance for genome stability that repair DNA polymerases (Pols), such as Pols λ and β, which all exhibit considerably reduced fidelity when replicating undamaged DNA, are tightly regulated, because their misregulation could lead to mutagenesis. Recently, we found that the correct repair of the abundant and highly miscoding oxidative DNA lesion 7,8-dihydro-8-oxo-2'-deoxyguanine (8-oxo-G) is performed by an accurate repair pathway that is coordinated by the MutY glycosylase homologue (MutYH) and Pol λ in vitro and in vivo. Pol λ is phosphorylated by Cdk2/cyclinA in late S and G2 phases of the cell cycle, promoting Pol λ stability by preventing it from being targeted for proteasomal degradation by ubiquitination. However, it has remained a mystery how the levels of Pol λ are controlled, how phosphorylation promotes its stability, and how the engagement of Pol λ in active repair complexes is coordinated. Here, we show that the E3 ligase Mule mediates the degradation of Pol λ and that the control of Pol λ levels by Mule has functional consequences for the ability of mammalian cells to deal with 8-oxo-G lesions. Furthermore, we demonstrate that phosphorylation of Pol λ by Cdk2/cyclinA counteracts its Mule-mediated degradation by promoting recruitment of Pol λ to chromatin into active 8-oxo-G repair complexes through an increase in Pol λ's affinity to chromatin-bound MutYH. Finally, MutYH appears to promote the stability of Pol λ by binding it to chromatin. In contrast, Pol λ not engaged in active repair on chromatin is subject for proteasomal degradation.     

Regulation of pancreatic islet cell survival and replication by γ-aminobutyric acid

Aims/hypothesis Pancreatic islets have evolved remarkable, though poorly understood mechanisms to modify beta cell mass when nutrient intake fluctuates or cells are damaged. We hypothesised that appropriate and timely adjustments in cell number occur because beta cells release proliferative signals to surrounding cells when stimulated by nutrients and ‘bleed’ these growth factors upon injury. Materials and methods In rat pancreatic islets, we measured DNA content, insulin content, insulin secretion after treatment, immunoblots of apoptotic proteins and the uptake of nucleoside analogues to assess the ability of γ-aminobutyric acid (GABA), which is highly concentrated in beta cells, to act as a growth and survival factor. This focus is supported by work from others demonstrating that GABA increases cell proliferation in the developing nervous system, acts as a survival factor for differentiated neurons and, interestingly, protects plants under stress. Results Our results show that DNA, insulin content and insulin secretion are higher in freshly isolated islets treated with GABA or GABA B receptor agonists. Exposure to GABA upregulated the anti-apoptotic protein B-cell chronic lymphocytic leukaemia XL and limited activation of caspase 3 in islets. The cellular proliferation rate in GABA-treated islets was twice that of untreated controls. Conclusions/interpretation We conclude that GABA serves diverse purposes in the islet, meeting a number of functional criteria to act as an endogenous co-regulator of beta cell mass.     

Regulation of pancreatic β-cell survival by nitric oxide: clinical relevance

The reduction of pancreatic β-cell mass is an important factor in the development of type 1 and type 2 diabetes. Understanding the mechanisms that regulate the maintenance of pancreatic β-cell mass as well as β-cell death is necessary for the establishment of therapeutic strategies. In this context, nitric oxide (NO) is a diatomic, gaseous, highly reactive molecule with biological activity that participates in the regulation of pancreatic β-cell mass. Two types of cellular responses can be distinguished depending on the level of NO production. First, pancreatic β-cells exposed to inflammatory cytokines, lipid stress or hyperglycaemia produce high concentrations of NO, mainly due to the activation of inducible NO synthase (iNOS), thus promoting cell death. Meanwhile, under homeostatic conditions, low concentrations of NO, constitutively produced by endothelial NO synthase (eNOS), promote cell survival. Here, we will discuss the current knowledge of the NO-dependent mechanisms activated during cellular responses, emphasizing those related to the regulation of cell survival.     

Regulation of rat ileal water absorption by central α2 adrenergic receptors

This study examined the role of central adrenergic receptors in the regulation of water absorption by the rat ileum. Clonidine, an ₂ -adrenergic receptor agonist, increased water absorptionin vivo following intracerebroventricular but not intravenous injection. Intracerebroventricular injection of prazosin, an ₁ -adrenergic receptor antagonist, did not alter basal water absorption. Prazosin did not prevent the clonidine effect. In contrast, the ₂ -adrenergic receptor antagonist, yohimbine, reduced basal absorption and prevented the intracerebroventricular clonidine effect. Treatment with reserpine reduced ileal norepinephrine content by 98%, reduced basal water absorption consistent with a loss of sympathetic outflow to the mucosa, but did not prevent the increase in water absorption due to intracerebroventricular clonidine. These results suggest that central ₂ receptors regulate water absorption by the rat ileum. The clonidine-induced increase in water absorption is not mediated by the sympathetic nerves innervating the intestine.This work was supported by a grant from the Research Service of the Veteran's Administration, Washington, D.C.This work was presented in part at the American Gastroenterological Association Meeting in San Francisco, California, and has been published in abstract form (Gastroenterology 90:1418, 1986).     

Regulation of blood–testis barrier dynamics by TGF-β3 is a Cdc42-dependent protein trafficking event

In the testis, the blood–testis barrier (BTB) is constituted by specialized junctions between adjacent Sertoli cells in the seminiferous epithelium near the basement membrane. Although the BTB is one of the tightest blood–tissue barriers in the mammalian body, it undergoes extensive restructuring at stage VIII of the seminiferous epithelial cycle to facilitate the transit of preleptotene spermatocytes. Thus, meiosis and postmeiotic germ cell development take place in the seminiferous epithelium behind the BTB. Cytokines (e.g., TGF-β3) are known to regulate BTB dynamics by enhancing the endocytosis of integral membrane proteins and their intracellular degradation. This thus reduces the levels of proteins above the spermatocytes in transit at the BTB, causing its disruption after testosterone-induced new tight junction (TJ) fibrils are formed behind these cells. By using Sertoli cells cultured in vitro with an established TJ permeability barrier that mimicked the BTB in vivo, Cdc42 was shown to be a crucial regulator that mediated the TGF-β3–induced BTB disruption. TGF-β3 was shown to activate Cdc42 to its active GTP-bound form. However, an inactivation of Cdc42 by overexpressing its dominant-negative mutant T17N in Sertoli cell epithelium was shown to block the TGF-β3–induced acceleration in protein endocytosis. Consequently, this prevented the disruption of Sertoli cell TJ permeability barrier and redistribution of TJ proteins (e.g., CAR and ZO-1) from the cell–cell interface to cell cytosol caused by TGF-β3. In summary, Cdc42 is a crucial regulatory component in the TGF-β3–mediated cascade of events that leads to the disruption of the TJ fibrils above the preleptotene spermatocytes to facilitate their transit.     

Regulation of cholesterol biosynthetic pathway in patients with the Smith–Lemli–Opitz syndrome

The Smith–Lemli–Opitz syndrome (SLOS) is a recessively inherited birth disorder caused by a defect in 7-dehydrocholesterol (3-hydroxysteroid) ⁷-reductase, the final enzyme in cholesterol biosynthesis. To investigate in vivo regulation of the cholesterol biosynthetic pathway in SLOS, we measured hepatic microsomal sterol concentrations and activities of several key enzymes in the pathway, including HMG-CoA synthase, HMG-CoA reductase, squalene synthase and 7-dehydrocholesterol ⁷-reductase in liver specimens from a patient with SLOS and 11 controls. Hepatic microsomal 7-dehydrocholesterol ⁷-reductase activity in the patient was less than 1% of the control mean, and decreased cholesterol concentration and markedly increased 7- and 8-dehydrocholesterol concentrations were observed in the patient's microsomes. HMG-CoA synthase and squalene synthase activities in the patient were upregulated to 149% and 532%, respectively, while the activity of HMG-CoA reductase, the rate-limiting enzyme in the pathway, was reduced to 39% of the control mean. Downregulation of HMG-CoA reductase activity in SLOS was supported by measuring plasma levels of mevalonic acid, the immediate product of HMG-CoA reductase. The levels in SLOS patients (n=9) were significantly low compared with age-matched controls (n=8) (12±2 vs 28±6 nmol/L, p<0.05). These results suggest that in most SLOS patients in vivo HMG-CoA reductase is not stimulated in spite of blocked cholesterol biosynthetic pathway and reduced plasma and hepatic cholesterol concentrations.     

Regulation of Golgi structure and secretion by receptor-induced G protein βγ complex translocation

We show that receptor induced G protein βγ subunit translocation from the plasma membrane to the Golgi allows a receptor to initiate fragmentation and regulate secretion. A lung epithelial cell line, A549, was shown to contain an endogenous translocating G protein γ subunit and exhibit receptor-induced Golgi fragmentation. Receptor-induced Golgi fragmentation was inhibited by a shRNA specific to the endogenous translocating γ subunit. A kinase defective protein kinase D and a phospholipase C β inhibitor blocked receptor-induced Golgi fragmentation, suggesting a role for this process in secretion. Consistent with βγ translocation dependence, fragmentation induced by receptor activation was inhibited by a dominant negative nontranslocating γ3. Insulin secretion was shown to be induced by muscarinic receptor activation in a pancreatic β cell line, NIT-1. Induction of insulin secretion was also inhibited by the dominant negative γ3 subunit consistent with the Golgi fragmentation induced by βγ complex translocation playing a role in secretion.     

Regulation of cell–matrix contacts and β-catenin signaling in VSMC by integrin-linked kinase: implications for intimal thickening

Vascular smooth muscle cell (VSMC) proliferation and migration is responsible for intimal thickening that occurs in restenosis and atherosclerosis. Integrin-linked kinase (ILK) is a serine/threonine protein kinase implicated in signaling pathways involved in cell proliferation and migration. We studied the involvement of ILK in intimal thickening. ILK expression was significantly increased in two models of intimal thickening: balloon-injured rat carotid arteries and human saphenous vein organ cultures. Over-expression of a dominant negative ILK (DN-ILK) significantly reduced intimal thickening by approximately 50% in human saphenous vein organ cultures, demonstrating an important role in intimal thickening. ILK protein and activity was reduced on laminin and up-regulated on fibronectin, indicating ILK protein expression is modulated by extracellular matrix composition. Inhibition of ILK by siRNA knockdown and DN-ILK significantly decreased VSMC proliferation and migration while wild type ILK significantly increased proliferation and migration on laminin, confirming an essential role of ILK in both processes. Localization of paxillin and vinculin and protein levels of FAK and phospho-FAK indicated that inhibition of ILK reduced focal adhesion formation. Additionally, inhibition of ILK significantly attenuated the presence of the cell-cell complex proteins N-cadherin and beta-catenin, and beta-catenin signaling. We therefore suggest ILK modulates VSMC proliferation and migration at least in part by acting as a molecular scaffold in focal adhesions as well as modulating the stability of cell-cell contact proteins and beta-catenin signaling. In summary, ILK plays an important role in intimal thickening by modulating VSMC proliferation and migration via regulation of cell-matrix and cell-cell contacts and beta-catenin signaling.     

Regulation of glucagon secretion by glucose: paracrine, intrinsic or both?

Glucagon secretion is regulated by glucose but the mechanisms involved remain hotly debated. Both intrinsic (within the α-cell itself) and paracrine (mediated by factors released β- and/or δ-cells) have been postulated. Glucagon secretion is maximally suppressed by glucose concentrations that do not affect insulin and somatostatin secretion, a finding that highlights the significance of intrinsic regulation of glucagon secretion. Experiments on islets from mice lacking functional ATP-sensitive potassium channels (K(ATP)-channels) indicate that these channels are critical to the α-cell's capacity to sense changes in extracellular glucose. Here, we review recent data on the intrinsic and paracrine regulation of glucagon secretion in human pancreatic islets. We propose that glucose-induced closure of the K(ATP)-channels, via membrane depolarization, culminates in reduced electrical activity and glucagon secretion by voltage-dependent inactivation of the ion channels involved in action potential firing. We further demonstrate that glucagon secretion measured in islets isolated from donors with type-2 diabetes is reduced at low glucose and that glucose stimulates rather than inhibits secretion in these islets. We finally discuss the relative significance of paracrine and intrinsic regulation in the fed and fasted states and propose a unifying model for the regulation of glucagon secretion that incorporates both modes of control.