Critical roles of isoleucine-364 and adjacent residues in a hydrophobic gate control of phospholipid transport by the mammalian P4-ATPase ATP8A2

P4-ATPases (flippases) translocate specific phospholipids such as phosphatidylserine from the exoplasmic leaflet of the cell membrane to the cytosolic leaflet, upholding an essential membrane asymmetry. The mechanism of flipping this giant substrate has remained an enigma. We have investigated the importance of amino acid residues in transmembrane segment M4 of mammalian P4-ATPase ATP8A2 by mutagenesis. In the related ion pumps Na⁺,K⁺-ATPase and Ca²⁺-ATPase, M4 moves during the enzyme cycle, carrying along the ion bound to a glutamate. In ATP8A2, the corresponding residue is an isoleucine, which recently was found mutated in patients with cerebellar ataxia, mental retardation, and dysequilibrium syndrome. Our analyses of the lipid substrate concentration dependence of the overall and partial reactions of the enzyme cycle in mutants indicate that, during the transport across the membrane, the phosphatidylserine head group passes near isoleucine-364 (I364) and that I364 is critical to the release of the transported lipid into the cytosolic leaflet. Another M4 residue, N359, is involved in recognition of the lipid substrate on the exoplasmic side. Our functional studies are supported by structural homology modeling and molecular dynamics simulations, suggesting that I364 and adjacent hydrophobic residues function as a hydrophobic gate that separates the entry and exit sites of the lipid and directs sequential formation and annihilation of water-filled cavities, thereby enabling transport of the hydrophilic phospholipid head group in a groove outlined by the transmembrane segments M1, M2, M4, and M6, with the hydrocarbon chains following passively, still in the membrane lipid phase.Members of the P4 subfamily of P-type ATPases are known as flippases, because they transport (flip) specific phospholipids from the exoplasmic to the cytoplasmic leaflet of the plasma membrane bilayer (1–3). Thus, they establish and maintain a physiologically essential asymmetry between the two leaflets, with phosphatidylserine (PS) and phosphatidylethanolamine (PE) being concentrated in the cytoplasmic leaflet and phosphatidylcholine (PC) and sphingomyelin in the exoplasmic leaflet. There are 14 human P4-ATPases, and mutations in many of these are linked to severe disorders (4–7). On the basis of amino acid sequence alignment, the P4-ATPases are predicted to structurally resemble the classic P-type ATPase cation pumps Na⁺,K⁺-ATPase and Ca²⁺-ATPase, possessing a transmembrane domain with 10 helices (M1–M10) and three cytoplasmic domains, P (phosphorylation), N (nucleotide binding), and A (actuator) (Fig. 1A), known from crystal structures to undergo large movements during the catalytic cycle (8, 9). Recently, we showed that the P4-ATPase ATP8A2, like the cation pumps, forms an aspartyl-phosphorylated intermediate and undergoes a catalytic cycle involving the conformations E₁, E₁P, E₂P, and E₂ similar to the Post-Albers scheme originally proposed for the Na⁺,K⁺-ATPase (Fig. 1 B and C) (10, 11). We found that the dephosphorylation of E₂P is activated by the transported substrates PS and PE, similar to K⁺ activation of dephosphorylation in Na⁺,K⁺-ATPase (Fig. 1C), and that K873, located in transmembrane helix M5 of ATP8A2 at a position equivalent to that of a K⁺-binding serine in Na⁺,K⁺-ATPase, is critical for the sensitivity of the phosphoenzyme to PS and PE (12), which seems to argue for a high degree of mechanistic similarity of the P4-ATPases to Na⁺,K⁺-ATPase and Ca²⁺-ATPase, not only in relation to the catalysis of ATP hydrolysis, but also in the transduction of the liberated energy into substrate movement. The M5 lysine might function directly or indirectly in binding of the phospholipid (12). It is, however, an open question how the flippase is able to move a phospholipid, which is ∼10-fold larger than the ions transported by Na⁺,K⁺-ATPase and Ca²⁺-ATPase and which has to reorient during the translocation. This enigma has been referred to as the “giant substrate problem” (13, 14). On the other hand, it is now clear that no protein other than the P4-ATPase catalytic chain in association with its small CDC50 subunit (β-subunit) is required for the flippase function, because the flipping of specific phospholipids is retained after purification and reconstitution of these proteins in lipid vesicles (2, 3, 12). In Na⁺,K⁺-ATPase and Ca²⁺-ATPase, the central helices, M4, M5, and M6, play major roles in cation binding and occlusion. However, in recent studies using mutagenesis to exchange residues between the two yeast flippases Dnf1 and Drs2 of differing phospholipid head group specificity, side-chains critical to the PS specificity of Drs2 have been pinpointed at two interfacial regions flanking transmembrane helices M1–M3, outside the central core region of the protein, thus leading to the suggestion that the transport pathway for the lipid is unique compared with the canonical pathway used by the P-type cation pumps (13, 15). From these results, it also appears that a tyrosine at the cytoplasmic border of M4 (not present in Drs2 or mammalian PS flippases) participates in selection against PS in Dnf1 (13, 16).Open in a separate windowFig. 1.Topology and reaction schemes of flippase and Na⁺,K⁺-ATPase. (A) Diagram of P4-ATPase topology indicating P-type ATPase domains and residues studied here by mutagenesis, as well as K873 of M5 studied previously (12). (B) Proposed flippase reaction cycle. (C) Reaction cycle of Na⁺,K⁺-ATPase [Post-Albers scheme (10, 11)]. E₁, E₁P, E₂P, and E₂ are the major enzyme forms, the P indicating phosphorylation.Transmembrane segment M4 is a key element in the mechanism of the cation-transporting P-type ATPases. In the well-documented catalytic cycles of Na⁺,K⁺-ATPase and Ca²⁺-ATPase, the glutamate of M4 binds Na⁺/K⁺ and Ca²⁺/H⁺ and is alternately exposed to the two sides of the membrane during the cycle (9, 17, 18). Mutations of nearby located residues in M4 have been shown to block the E₁P→E₂P or E₂P→E₂ transition (19–21). Crystal structures of the Ca²⁺-ATPase indicate that the E₁P→E₂P conformational change involves a vertical movement of M4, like a pump rod, corresponding to a whole turn of the helix, allowing delivery of Ca²⁺ bound at the M4 glutamate to the lumen. During the E₂→E₁ transition of the dephosphoenzyme, M4 moves a similar distance in the opposite direction toward the cytosol (8, 9). In ATP8A2, the residue located at the position equivalent to the ion binding glutamate in M4 of Ca²⁺-ATPase and Na⁺,K⁺-ATPase is an isoleucine, which is highly conserved among P4-ATPases (Fig. S1). Intriguingly, a missense mutation of this isoleucine to methionine was recently identified as the cause of cerebellar ataxia, mental retardation, and dysequilibrium (CAMRQ) syndrome in a Turkish family (22).Here, we investigated the functional consequences of mutations of isoleucine-364 (I364) and adjacent residues of ATP8A2 (see Fig. 1A for overview). The specific interaction with phospholipids and the individual partial reactions of the enzyme cycle were studied, and we provide evidence that I364 is crucial to the lipid transport, with mutations affecting the affinity for the phospholipid or the dissociation of the translocated lipid toward the cytoplasmic leaflet. In addition, N359 of M4 appears to play an important functional role. Molecular modeling suggests a mechanistic explanation of our observations, showing a remarkable movement of large, water-filled cavities during the transition between the E₂P and E₂ conformations. Moreover, the water flow appears to be gated by a central hydrophobic cluster composed of the side-chains of I364 and nearby conserved hydrophobic residues of M1, M2, and M6. Among these, I115 of M2 is found to be as mutation sensitive as I364 and N359. These data suggest a unique water-mediated transport pathway for the phospholipid head group along a groove outlined by M1, M2, M4, and M6, which could allow the lipid tail to follow passively, still in the membrane lipid phase, jutting out between M2 and M6. In such a mechanism, movement of M4 and in particular I364 would be as crucial to energy transduction and movement of the transported substrate as in the cation-translocating P-type ATPases.     

Enhanced Mg2+-ATPase activity in ghosts from HS erythrocytes and in normal ghosts stripped of membrane skeletal proteins may reflect enhanced aminophospholipid translocase activity

Hereditary spherocytosis (HS) is a congenital haemolytic anaemia which is characterized by a great variety of structural defects in the red cell's membrane skeleton and/or deficiencies in particular membrane (skeletal) proteins. Enhanced (Mg²⁺)-dependent adenosine triphosphatase (Mg²⁺-ATPase) activities, varying from 115% to 160%, were invariably found in erythrocyte ghosts derived from 13 HS patients. Similarly, an enhancement of Mg²⁺-ATPase activity by 30% is observed in normal red cell ghosts that have been stripped of the greater part of their membrane skeletal proteins by treatment with a low ionic strength buffer. Reassociation of those stripped ghosts with spectrin reduces the enhanced Mg²⁺-ATPase activity to its original level. Since in both cases, HS ghosts and stripped normal ghosts, the stabilizing effects that the membrane skeleton exerts on the maintenance of an endofacial localization of the aminophospholipids are impaired, the enhanced Mg²⁺-ATPase activity is interpreted to reflect an increased activity of the aminophospholipid translocase. The present observations therefore support a role of the membrane skeleton in the stabilization of phospholipid asymmetry in the red cell membrane and consequently in reducing the energy consumption of the translocase.     

Conductance of P2X4 purinergic receptor is determined by conformational equilibrium in the transmembrane region

Ligand-gated ion channels are partially activated by their ligands, resulting in currents lower than the currents evoked by the physiological full agonists. In the case of P2X purinergic receptors, a cation-selective pore in the transmembrane region expands upon ATP binding to the extracellular ATP-binding site, and the currents evoked by α,β-methylene ATP are lower than the currents evoked by ATP. However, the mechanism underlying the partial activation of the P2X receptors is unknown although the crystal structures of zebrafish P2X₄ receptor in the apo and ATP-bound states are available. Here, we observed the NMR signals from M339 and M351, which were introduced in the transmembrane region, and the endogenous alanine and methionine residues of the zebrafish P2X₄ purinergic receptor in the apo, ATP-bound, and α,β-methylene ATP-bound states. Our NMR analyses revealed that, in the α,β-methylene ATP-bound state, M339, M351, and the residues that connect the ATP-binding site and the transmembrane region, M325 and A330, exist in conformational equilibrium between closed and open conformations, with slower exchange rates than the chemical shift difference (<100 s⁻¹), suggesting that the small population of the open conformation causes the partial activation in this state. Our NMR analyses also revealed that the transmembrane region adopts the open conformation in the state bound to the inhibitor trinitrophenyl-ATP, and thus the antagonism is due to the closure of ion pathways, except for the pore in the transmembrane region: i.e., the lateral cation access in the extracellular region.In chemical neurotransmission, various neurotransmitters bind to ligand-gated ion channels expressed in the plasma membrane of postsynaptic cells, such as the NMDA, AMPA, and P2X receptors, leading to changes in membrane potential and the concentration of intracellular ions. Each ligand for a ligand-gated ion channel has a distinct ability to evoke currents (1), and the ligands are classified according to the evoked current level: such as, full agonists, partial agonists, and antagonists. Partial agonists of ligand-gated ion channels reportedly offer clinical advantages over antagonists and full agonists in antidepressant and smoking-cessation treatment (2, 3).Two mechanisms have been proposed for the partial activation of the ligand-gated ion channels: the equilibrium between the open and closed conformations and the distinct conformation of the partial agonist-bound states from the closed and open conformations (4, 5). In the crystal structures of the extracellular region of the AMPA receptor, in which the distances between the two extracellular domains are changed upon agonist binding, the interdomain distances in the partial agonist-bound states correlated with the conductance level, suggesting that the AMPA receptor adopts specific intermediately permeable conformations (4, 6).The P2X receptors are a family of cation channels gated by extracellular ATP (1, 7–9) and are involved in many physiological and pathophysiological processes (10–12). Seven subtypes of the P2X receptors have been identified in mammals (13), and they share ∼40% sequence identity. The P2X₄ receptor is involved in the pathogenesis of chronic neuropathic, inflammatory pain and the endothelial cell-mediated control of vascular tone (11, 14, 15). Compared with ATP, α,β-methylene ATP (α,β-meATP), in which the oxygen atom linking the α- and β-phosphorous atoms of ATP is replaced by a methylene group (Fig. S1A), reportedly induces a lower maximum current in cells expressing the mouse, rat, and human P2X₄ receptors and other P2X receptors (16, 17).Open in a separate windowFig. S1.Characterization of the P2X₄ receptor. (A) Chemical structures of ATP and α,β-meATP. (B and C) TEVC recordings of ATP- and α,β-meATP-evoked currents from rat P2X₄ receptor expressed in Xenopus oocytes, respectively. In B, the currents were evoked twice by ATP (30 μM, 1 min, black bar). In C, the currents were firstly evoked by ATP (30 μM, 1 min, black bar) and subsequently by α,β-meATP (300 μM, 1 min, black bar). (D) TEVC recording of the ATP-evoked current (30 μM, 30 s, black bar) from the N-terminally EGFP-tagged ΔzfP2X₄–A′ construct expressed in Xenopus oocytes. (E) Size exclusion chromatogram of purified EGFP-tagged ΔzfP2X₄–A′ in rHDLs. Elution volumes corresponding to 17.0, 12.2, 10.4, and 7.1 nm Stokes diameters were determined by thyroglobulin, ferritin, catalase, and BSA, respectively. V₀ and 1CV are void volume and single column volume, respectively. (F) SDS/PAGE analyses of purified ΔzfP2X₄–A′ embedded in rHDLs. The samples were analyzed by 12% SDS/PAGE with Coomassie Brilliant Blue staining. (G) Measurement of [³H]ATP saturation binding to the purified ΔzfP2X₄–A′ in rHDLs. (H and I) Estimation of the effects of deuteration based on the crystal structures of zfP2X₄ (PDB ID code 4DW1) and the deuteration incorporation rates. The plots on the Left (without deuteration) and the Right (with deuteration) are the sums of the inverse sixth power of the distances between pseudoatoms centered on the methyl hydrogens of M108, M249, M268, or M325 and each hydrogen atom in the crystal structure of zfP2X₄ (sums of the r⁻⁶) and the sums of the r⁻⁶ multiplied by [1 − (deuterium incorporation rates)] of each hydrogen atom, respectively. The graphs in H and I were calculated from the crystal structure in the apo state (PDB ID code 4DW0) and that in the ATP-bound state (PDB ID code 4DW1), respectively. Sums of the r⁻⁶ of each methionine methyl group and Hαβγ of the intraresidue methionine (green), Hαβγ of the interresidue methionine (light green), Hαβ of tyrosine (light violet), Hδεζη of tryptophan (orange), Hαβδεζ of phenylalanine (pink), Hαβγ of valine (blue), Hαβγδ of leucine (light blue), Hαβγδ of isoleucine (cyan), Hαβγ of threonine (light cyan), Hαβ of alanine (red), Hαβγδ of arginine (dark blue), Hα of glycine (dark green), and Hαβ of serine (magenta) residues, and the other hydrogens connected to carbon atoms (other unexchangeable hydrogens, light gray) are shown with colors. Hydrogen atoms connected to nitrogen, oxygen, or sulfur atoms were not considered in these calculations because these hydrogens should be exchanged with deuterium in D₂O. The deuterium incorporation rates of the hydrogen atoms within each methionine residue (intraresidue) and the deuterium incorporation rates of other methionine residues (interresidue) were set to 98% and 85%, respectively, because the methionine residues would be derived from 85% of [α-, β-, γ-98% ²H-, methyl-¹³C]-methionine and 15% of nonlabeled methionine in the medium.The crystal structures of zebrafish P2X₄ receptor (zfP2X₄) (18, 19), together with mutational analyses (20–26), provided the structural basis for the channel opening of P2X receptors upon ATP binding. In the crystal structures, zfP2X₄ forms a homotrimer (27, 28), in which the transmembrane region of each subunit is composed of two helices (19). In the crystal structure of zfP2X₄ in the ATP-bound state, three ATP molecules are bound to the intersubunit nucleotide binding pockets. In addition, the region that connects the ATP-binding site and the transmembrane region, which is referred to as the “lower body” (Fig. 1 A and B), is expanded by ∼10 Å in the ATP-bound state, and a pore is formed in the transmembrane region, which is proposed to expand by the iris-like movement of the transmembrane helices (18). However, the mechanism underlying the partial activation of P2X receptors is unknown because the structures of the P2X receptors have not been examined in the partial agonist-bound states.Open in a separate windowFig. 1.NMR resonances from the endogenous methionine residues of zfP2X₄ in rHDL. (A and B) Distribution of the methionine residues in the ΔzfP2X₄–A′. One subunit from the crystal structure of zfP2X₄ in the apo form (A) (PDB ID code 4DW0) and one from the ATP-bound form (B) (PDB ID code 4DW1) are shown in ribbons. The lower body and the right flipper are yellow. The A330 residues, the methionine residues, and the residues in which methionine mutations were introduced, L339 and L351, are depicted by green sticks. ATP is depicted by red sticks. Dummy atoms generated by Orientations of Proteins in Membranes (OPM), which represent membrane boundary planes, are gray. (C) Overlaid ¹H-¹³C HMQC spectra of [²H-11AA, α, β-²H, methyl-¹³C-Met]ΔzfP2X₄-A′, embedded in rHDLs, in the apo state (black) and the ATP-bound state (red). The regions with resonances from methionine residues are shown, and the assigned resonances are indicated. The centers of the resonances are indicated with dots. Cross-sections at lines through the centers of each resonance in the ATP-bound state and the cross-sections of the spectra using [α, β-²H, methyl-¹³C-Met]ΔzfP2X₄-A′ are shown on the top of the overlaid spectra. The intensities of the cross-sections were normalized by the concentration of ΔzfP2X₄-A′ and the conditions of the NMR measurements.The P2X₄ receptor used in the previous crystallographic studies was solubilized by detergents, which are widely used for structural investigations of membrane proteins, but the P2X₄ receptor is embedded in lipid bilayers under physiological conditions. It was recently reported that reconstituted high-density lipoproteins (rHDLs), which are also known as nanodiscs (29), can accommodate membrane proteins within a 10-nm-diameter disk-shaped lipid bilayer (30). The rHDLs reportedly provide a lipid environment with more native-like properties, compared with liposomes, in terms of the lateral pressure and curvature profiles because detergent micelles have strong curvature and different lateral pressure profiles from lipid membranes (31). Our NMR analyses of a G protein-coupled receptor (GPCR) and an ion channel in rHDL lipid bilayers revealed that the population and the exchange rates of the conformational equilibrium determine their signal transduction and ion transport activities (32–34) and that the population of the active conformation of the GPCR in rHDLs correlated better with the signaling levels than that in detergent micelles (32). Therefore, NMR investigations of membrane proteins in the lipid bilayer environments of rHDLs are necessary for accurate measurements of the exchange rates and the populations in conformational equilibrium.Here, we used NMR to observe the conformational equilibrium of the alanine and methionine residues of zfP2X₄ bound to α,β-meATP in rHDLs. Based on the conformational equilibrium, we discuss the mechanism underlying the partial activation of P2X receptors.     

Cloning and expression of SLC10A4, a putative organic anion transport protein

INTRODUCTION Bile acids, the major solute in bile, are physiologically important for promoting bile flow and facilitating the absorption of dietary lipids[1,2]. Bile acids are also involved in cholesterol homeostasis, xenobiotic excretion, as well as apop…     

Band 3 (AE1, SLC4A1)-mediated transport of stilbenedisulfonates. III: Role of solute and protein structure in proton-activated stilbenedisulfonate influx

DBDS (4,4'-dibenzamido-2,2'-stilbenedisulfonate) influx into magnesium resealed ghosts (MRSG) occurs over the anion/proton co-transport pH range (pK approximately 5.0). Here, factors are studied which may influence the pH dependence of DBDS transport. Accumulation of various stilbenedisulfonate (SD) molecules was studied and found to be correlated with the hydrophobicity of the R-groups (Hansch factor), not protonation of the sulfonates. The role of proton binding to glutamate 681 was found not to be part of the rate-limiting step in DBDS uptake by MRSG. Finally, the pH dependence of changes in quaternary structure/conformational state was investigated using an assay involving photo-crosslinking of band 3 subunits in the presence of DASD (4,4'-diazido-2,2'-stilbenedisulfonate). Lowering the pH promoted intersubunit crosslinking by DASD, with a pK value of 4.75+/-1.0. This value is comparable to the pK for DBDS binding to the "second" class of sites on control band 3 (pK = 5.01+/-0.16), and to DBDS influx into control MRSG (pK values between 4.57+/-0.15 and 4.7+/-0.1). Susceptibility to photo-crosslinking was reversed by raising the pH prior to initiation of the reaction. Significantly, no photo-crosslinking was observed between pH 6.0 and 8.0, where band 3 subunits are known to exist as stable dimers and tetramers. We conclude that intersubunit photo-crosslinking does not simply involve random collision between photo-activated DASD and band 3. Rather, proton binding to band 3 either alters the conformation at the interface between subunits of pre-existing tetramers, or it promotes self-association of stable dimers to a "novel" tetrameric conformational state.     

Band 3 (AE1, SLC4A1)-mediated transport of stilbenedisulfonates. II: Evidence for transmembrane allosteric interactions between the "primary" stilbenedisulfonate binding site and the stilbenedisulfonate efflux site

Results from the first paper in this series indicated that the "primary" stilbenedisulfonate (PSD) site was not located on the DBDS (4, 4'-dibenzamido-2, 2'-stilbenedisulfonate) transport pathway into magnesium resealed ghosts (MRSG). Rather, transport correlated with DBDS binding to the "second" class of proton-activated binding sites located on the membrane domain of band 3 [Biochem. J. 388 (2005) 343]. Here we report the discovery that reversible binding of extracellular H2DIDS (4, 4'-diisothiocyanatodihydro-2, 2'-stilbenedisulfonate) to the PSD site causes a greater than 5-fold acceleration in the rate of DBDS efflux from pre-loaded MRSG at physiological pH. Pre-labeling all of the PSD sites with H2DIDS inhibited the acceleration effect completely, thus confirming mediation by band 3. Acceleration of DBDS efflux could be mimicked by establishing an externally directed proton gradient (acidic inside, pH 7.4 outside). Under these conditions, addition of extracellular H2DIDS neither accelerated DBDS efflux further nor was proton-induced acceleration inhibited. The results of this paper support the view that the PSD binding site on band 3 is an allosteric regulatory site which is not located on the SD transport pathway. We propose a model where H2DIDS binding to the PSD site modulates activity at the "second" class of sites by raising the pK for transport of DBDS into the physiological pH range.     

A possible mechanism of increased sodium influx in red cells with abnormal membrane lipid levels induced by phospholipase A2

Membrane transport--sodium (Na+) influx and calcium (Ca2+) uptake--was examined in human mature red cells treated with phospholipase A2 (PLase A2) from snake venom. PLase A2-induced conversion of phosphatidyl choline (PC) to lysophosphatidyl choline (L-PC) was associated with a marked increase in Na+ influx and Ca2+ uptake. After L-PC was removed from the cell membrane of the PLase A2-treated red cells in the presence of albumin, an additional increase in Ca2+ transport was observed. These results indicate that membrane lipid abnormalities, such as increased L-PC and/or a loss of total lipids, appear to induce increased membrane transport.     

Critical role of aquaporin-4 (AQP4) in astrocytic Ca2+ signaling events elicited by cerebral edema

Aquaporin-4 (AQP4) is a primary influx route for water during brain edema formation. Here, we provide evidence that brain swelling triggers Ca(2+) signaling in astrocytes and that deletion of the Aqp4 gene markedly interferes with these events. Using in vivo two-photon imaging, we show that hypoosmotic stress (20% reduction in osmolarity) initiates astrocytic Ca(2+) spikes and that deletion of Aqp4 reduces these signals. The Ca(2+) signals are partly dependent on activation of P2 purinergic receptors, which was judged from the effects of appropriate antagonists applied to cortical slices. Supporting the involvement of purinergic signaling, osmotic stress was found to induce ATP release from cultured astrocytes in an AQP4-dependent manner. Our results suggest that AQP4 not only serves as an influx route for water but also is critical for initiating downstream signaling events that may affect and potentially exacerbate the pathological outcome in clinical conditions associated with brain edema.     

Evidence for a role of free radicals by synthesized scavenger, 2-octadecylascorbic acid,in cerulein-induced mouse acute pancreatitis

To define the role of free radicals and of lipid peroxide involvement during the progress of cerulein-induced acute pancreatitis in mice, we evaluated the effect of a novel free radical scavenger, 2-octadecylascorbic acid (CV-3611), on pancreatic edema formation, and the levels of serum enzymes (amylase, lipase) and of lipid peroxide in pancreatic tissue. Mice were divided into three groups: control group, intraperitoneal injection of saline only; pancreatitis group, cerulein 50 g/kg injected intraperitoneally six times at 1-hr intervals; treatment group, CV-3611 10 mg/kg subcutaneously just after intraperitoneal cerulein injection. After the cerulein injection, the degree of pancreatic edema formation, serum amylase and lipase levels, and the amount of lipid peroxide in pancreatic tissue increased significantly during the observation period of 12 hr. Treatment with CV-3611 resulted in significant reduction in pancreatic edema formation at 3.5 hr (P<0.05) and 9 hr (P<0.05), serum amylase and lipase levels at 3.5 hr (P<0.05) and 12 hr (P<0.05), and lipid peroxide levels at 3.5 hr (P<0.05), 6 hr (P<0.05) and 12 hr (P<0.05). These results indicate that a novel free radical scavenger, CV-3611, has a strong therapeutic effect during the development of acute pancreatitis and suggest that oxygenderived free radicals play an important role in the pathogenesis of acute pancreatitis.     

Stimulation of 2-deoxyglucose uptake in rat adipocytes by a human growth hormone fragment (hGH 4-15)

Summary The in vivo and in vitro effects of a hypoglycaemic fragment of human growth hormone containing the sequence H₂N-Ile-Pro- Leu-Ser-Arg-Leu-Phe-Asp-Asn-Ala-Met-Leu-COOH (hGH 4–15) on 2-deoxy-[1-¹⁴C]-D-glucose uptake in adipocytes were studied. The isolated cells from rats after a single intravenous injection of hGH 4–15 (1 mg/kg) significantly increased uptake of 2-deoxyglucose (p < 0.005). Adipocytes from untreated rats pre-incubated with the hGH fragment (10 g/ml) at 37°C for 30 min also clearly showed an elevated uptake of 2-deoxyglucose in the absence and the presence of exogenous insulin. The effect of hGH 4–15 was concentration-dependent, steadily increasing with the maximum effect at 10 g/ml. The present findings suggest that the enhancement of glucose transport in target tissues may be a major contributing factor to the hypoglycaemic action of the amino-terminal fragments of human growth hormone.     

A role for altered TLR gene expression in association with increased expression of CD200R in the induction of mucosal tissue CD4(+) Treg in aged mice following gavage with a liver extract along with intramuscular monophosphoryl lipid A (MPLA) injection

Previous studies showed a fetal sheep liver extract (FSLE), in association with LPS, injected into aged (>20 months) mice reversed the altered polarization (increased IL-4 and IL-10 with decreased IL-2 and IFN-gamma) in cytokine production seen from ConA stimulated lymphoid cells of those mice. Aged mice show a >60% decline in numbers and suppressive function of both CD4(+)CD25(+)Foxp3(+)Treg and so-called Tr3 (CD4(+)TGFbeta(+)). Their number/function is restored to levels seen in control (8-week-old) mice by FSLE. We have reported at length on the ability of a novel pair of immunoregulatory molecules, members of the TREM family, namely CD200:CD200R, to control development of dendritic cells (DCs) which themselves regulate production of Foxp3(+) Treg. The latter express a distinct subset of TLRs which control their function. We report that a feature of the altered Treg expression following combined treatment with FSLE and monophosphoryl lipid A, MPLA (a bioactive component of lipid A of LPS) is the altered gene expression both of distinct subsets of TLRs and of CD200Rs. We speculate that this may represent one of the mechanisms by which FSLE and MPLA alter immunity in aged mice.     

Anti-islet autoantibodies detected by monoclonal antibody 1A2: further studies suggesting a role in the pathogenesis of IDDM

Summary Insulin-dependent diabetes mellitus (IDDM) is associated with autoantibodies to several pancreatic islet antigens. We have described an assay in which autoantibodies displace a radiolabelled monoclonal anti-islet antibody. Sera from 87 % of 429 children at time of diagnosis of IDDM were positive, while sera from control groups had much lower prevalences (1.3–19 %). Sera from 41.9 % of diabetic subjects remained positive after 20 years duration of IDDM. Sera from 23.6 % of parents and 37.9 % of non-diabetic siblings were positive. Twenty relatives who subsequently developed IDDM had the same prevalence of the antibodies (85 %) as did the patients at time of diagnosis. These findings confirm that the autoantibodies detected by monoclonal antibody (mAb) 1A2 are common at the onset of IDDM and their presence prior to the onset of hyperglycaemia suggests that this method may be useful in screening non-diabetic populations. The high prevalence of antibodies in relatives reduces the efficacy for diabetes prediction, but suggests either that generation of these antibodies is an autosomal dominant trait, or that the antigen detected by these antibodies is cross-reactive with a common environmental antigen. Differentiation between these hypotheses will await the identification of the specific islet-cell antigen detected by mAb 1A2. [Diabetologia (1996) 39: 1365–1371] Received: 15 February 1996 and in final revised form: 10 June 1996     

Role of premixed insulin analogues in the treatment of patients with type 2 diabetes mellitus: A narrative review (预混胰岛素类似物在2型糖尿病治疗中的作用：叙述性综述)

Because of the progressive nature of type 2 diabetes mellitus (T2DM), insulin therapy will eventually become necessary in most patients. Recent evidence suggests that maintaining optimal glycemic control by early insulin therapy can reduce the risk of microvascular and macrovascular complications in patients with T2DM. The present review focuses on relevant clinical evidence supporting the use of premixed insulin analogues in T2DM when intensifying therapy, and as starter insulins in insulin‐naïve patients. Our aim is to provide relevant facts and clinical evidence useful in the decision‐making process of treatment selection and individualized treatment goal setting to obtain sustained blood glucose control.     

Current role of short‐term intensive insulin strategies in newly diagnosed type 2 diabetes (短期胰岛素强化治疗策略对初诊2型糖尿病患者的作用)

Type 2 diabetes mellitus (T2DM) is a progressive disease characterized by worsening insulin resistance and a decline in β‐cell function. Achieving good glycemic control becomes more challenging as β‐cell function continues to deteriorate throughout the disease process. The traditional management paradigm emphasizes a stepwise approach, and insulin has generally been reserved as a final armament. However, mounting evidence indicates that short‐term intensive insulin therapy used in the early stages of type 2 diabetes could improve β‐cell function, resulting in better glucose control and more extended glycemic remission than oral antidiabetic agents. Improvements in insulin sensitivity and lipid profile were also seen after the early initiation of short‐term intensive insulin therapy. Thus, administering short‐term intensive insulin therapy to patients with newly diagnosed T2DM has the potential to delay the natural process of this disease, and should be considered when clinicians initiate treatment. Although the early use of insulin is advocated by some guidelines, the optimal time to initiate insulin therapy is not clearly defined or easily recognized, and a pragmatic approach is lacking. Herein we summarize the current understanding of early intensive insulin therapy in patients with newly diagnosed T2DM, focusing on its clinical benefit and problems, as well as possible biological mechanisms of action, and discuss our perspective.