Glucose stimulates somatostatin secretion in pancreatic δ-cells by cAMP-dependent intracellular Ca2+ release

Somatostatin secretion from pancreatic islet δ-cells is stimulated by elevated glucose levels, but the underlying mechanisms have only partially been elucidated. Here we show that glucose-induced somatostatin secretion (GISS) involves both membrane potential-dependent and -independent pathways. Although glucose-induced electrical activity triggers somatostatin release, the sugar also stimulates GISS via a cAMP-dependent stimulation of CICR and exocytosis of somatostatin. The latter effect is more quantitatively important and in mouse islets depolarized by 70 mM extracellular K+, increasing glucose from 1 mM to 20 mM produced an ∼3.5-fold stimulation of somatostatin secretion, an effect that was mimicked by the application of the adenylyl cyclase activator forskolin. Inhibiting cAMP-dependent pathways with PKI or ESI-05, which inhibit PKA and exchange protein directly activated by cAMP 2 (Epac2), respectively, reduced glucose/forskolin-induced somatostatin secretion. Ryanodine produced a similar effect that was not additive to that of the PKA or Epac2 inhibitors. Intracellular application of cAMP produced a concentration-dependent stimulation of somatostatin exocytosis and elevation of cytoplasmic Ca2+ ([Ca2+]i). Both effects were inhibited by ESI-05 and thapsigargin (an inhibitor of SERCA). By contrast, inhibition of PKA suppressed δ-cell exocytosis without affecting [Ca2+]i. Simultaneous recordings of electrical activity and [Ca2+]i in δ-cells expressing the genetically encoded Ca2+ indicator GCaMP3 revealed that the majority of glucose-induced [Ca2+]i spikes did not correlate with δ-cell electrical activity but instead reflected Ca2+ release from the ER. These spontaneous [Ca2+]i spikes are resistant to PKI but sensitive to ESI-05 or thapsigargin. We propose that cAMP links an increase in plasma glucose to stimulation of somatostatin secretion by promoting CICR, thus evoking exocytosis of somatostatin-containing secretory vesicles in the δ-cell.

Electrophysiological properties of human β-cell lines EndoC-βH1 and -βH2 conform with human β-cells

The electrophysiological and secretory properties of the human β-cell lines EndoC-βH1 and EndoC-βH2 were investigated. Both cell lines respond to glucose (6-20mM) with 2-to 3-fold stimulation of insulin secretion, an effect that was mimicked by tolbutamide (0.2mM) and reversed by diazoxide (0.5mM). Glucose-induced insulin release correlated with an elevation of [Ca2+]i, membrane depolarization and increased action potential firing. KATP channel activity at 1mM glucose is low and increasing glucose to 6 or 20mM reduced KATP channel activity to the same extent as application of the KATP channel blocker tolbutamide (0.2mM). The upstroke of the action potentials in EndoC-βH1 and −βH2 cells observed at high glucose principally reflects activation of L- and P/Q-type Ca2+ channels with some small contribution of TTX-sensitive Na+ channels. Action potential repolarization involves activation of voltage-gated Kv2.2 channels and large-conductance Ca2+-activated K+ channels. Exocytosis (measured by measurements of membrane capacitance) was triggered by membrane depolarizations >10ms to membrane potentials above -30mV. Both cell lines were well-granulated (6,000-15,000 granules/cell) and granules consisted of a central insulin core surrounded by a clear halo. We conclude that the EndoC-βH1 and -βH2 cells share many features of primary human β-cells and that they represent a useful experimental model.

Inecalcitol Induces CD38 Expression in Multiple Myeloma and Myeloid Cell Lines

Targeting CD38 has proven to be an effective strategy for the treatment of relapsed refractory multiple myeloma (MM), with the recent approval of the first therapeutic monoclonal antibody directed against human CD38: daratumumab (Sanchez L et al., 2016, J Hematol Oncol; van de Donk NWC et al., 2016, Immunol Rev). Interestingly, the pretreatment levels of CD38 expression on MM cells were significantly higher in patients who achieved at least partial response (PR) as compared to patients who achieved less than PR (Nijhof IS et al., 2016, Blood). Therefore, increasing the levels of CD38 expression is the next logical pharmacological step to enhance the efficacy of anti-CD38 therapy. In this context, we tested inecalcitol (INE: 14epi-, 19nor-, 23yne-, 1,25dihydroxy-cholecalciferol) on the MM.1S cell line (from ATCC, CRL-2974) in culture and found a reproducible concentration-dependent increase in CD38 expression levels after 3 days of treatment in 4 independent experiments. CD38 levels were measured at the surface of the cells by flow cytometry (ACEA NovoCyte flow cytometer) after one hour of incubation at 4°C with a fluorescent anti-human CD38 mouse FITC antiboby (eBioscience, 11-0389) or with the matched isotype control mouse IgG1 K (eBioscience, 11-4714). More than a 2-fold increase in labelling intensity was already observed with 0.1 nM INE and a maximal 5-fold stimulation was achieved at 3 nM INE. The half-maximal effective concentration (EC50) of INE was 0.47 ± 0.23 nM (n=4). Next, we tested INE on L-363 cells, another MM cell line, purchased from DSMZ (ACC 49). In a single experiment, the L-363 cells were also shown to be responsive to INE, but about 10 times less sensitive than the MM.1S cells: a similar maximal 5-fold increase in CD38 levels on cell surface was also reached but at the much higher concentration of 30 nM INE and the threshold 2-fold stimulation of fluorescent labelling was obtained with 1 nM INE. The EC50 of INE in this single experiment was 4 nM. In normal cell culture conditions, both MM cell lines showed a mix of CD38-positive (CD38+) and CD38-negative (CD38-) cells, as illustrated by the distribution spectrum of intensities of individual cell labelling. Without any treatment by INE, the CD38+/CD38- ratio was higher among the MM.1s cells (80/20) than the L-363 cells (30/70). The effect of INE resulted in both a higher proportion of CD38+ cells (>93% CD38+ MM.1S cells treated from 0.3 nM and higher INE concentrations; only 58% CD38+ L-363 cells at 3 nM INE), and a higher mean intensity of individual cell labelling. Finally, we studied the HL-60 and U-937 cells classically described as representative of myeloid hematological malignancies. In normal culture conditions, both cell lines were CD38-. A concentration-dependent increase in CD38 expression levels on the surface of U-937 cells (from ATCC, CRL-1593,2) was obtained after 3 days of treatment with INE in the range of 0.1 to 10 nM in two independent experiments (EC50 = 0.47 nM and 0.76 nM). A concentration-dependent increase in CD38 expression levels on the surface of HL-60 cells (from ECACC, 98070106) was obtained after 3 days of treatment with INE in the range of 0.1 to 3 nM in two independent experiments (EC50s = 0.33 nM and 0.38 nM). All HL-60 or U-937 cells were CD38+ after 3 days of treatment with INE 0.3 nM or 1 nM, respectively. Inecalcitol is a vitamin D receptor agonist already characterized by a high anti-proliferative effect and a low calcemic potential (Okamoto R et al., 2012, Int J Cancer; Ma Y et al., 2013, Cell Cycle), allowing its administration at high oral doses to human cancer patients (Medioni J et al, 2014, Clin Cancer Res). The present findings suggest that INE could be tested in MM patients to increase the percentage of CD38+ MM cells and the density of CD38 at their surface, thereby potentially increasing the clinical response to anti-CD38 antibodies such as daratumumab. Furthermore, HL-60 and U-937 cells, all initially CD38-, became CD38+ after 3 days of in vitro treatment by INE, suggesting that CD38- myeloid hematological malignancies could potentially represent new indications for daratumumab when used after "priming" or in combination with INE. Disclosures Benjamin: Hybrigenics: Employment, Equity Ownership. Planquette:Hybrigenics: Employment, Equity Ownership. Delansorne:Hybrigenics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Inventor, but no royalties.

The UDP-diacylglucosamine Pyrophosphohydrolase LpxH in Lipid A Biosynthesis Utilizes Mn2+ Cluster for Catalysis

In Escherichia coli and the majority of β- and γ-proteobacteria, the fourth step of lipid A biosynthesis, i.e. cleavage of the pyrophosphate group of UDP-2,3-diacyl-GlcN, is carried out by LpxH. LpxH has been previously suggested to contain signature motifs found in the calcineurin-like phosphoesterase (CLP) family of metalloenzymes; however, it cleaves a pyrophosphate bond instead of a phosphoester bond, and its substrate contains nucleoside diphosphate moieties more common to the Nudix family rather than to the CLP family. Furthermore, the extent of biochemical data fails to demonstrate a significant level of metal activation in enzymatic assays, which is inconsistent with the behavior of a metalloenzyme. Here, we report cloning, purification, and detailed enzymatic characterization of Haemophilus influenzae LpxH (HiLpxH). HiLpxH shows over 600-fold stimulation of hydrolase activity in the presence of Mn2+. EPR studies reveal the presence of a Mn2+ cluster in LpxH. Finally, point mutants of residues in the conserved metal-binding motifs of the CLP family greatly inhibit HiLpxH activity, highlighting their importance in enzyme function. Contrary to previous analyses of LpxH, we find HiLpxH does not obey surface dilution kinetics. Overall, our work unambiguously establishes LpxH as a calcineurin-like phosphoesterase containing a Mn2+ cluster coordinated by conserved residues. These results set the scene for further structural investigation of the enzyme and for design of novel antibiotics targeting lipid A biosynthesis.

Effect of sialylation and complexity of FSH oligosaccharides on inhibin production by granulosa cells

Granulosa cell (GC) inhibin A and B production is regulated by FSH and gonadal factors. This gonadotrophin is released as a mixture of glycoforms, which induce different biological responses in vivo and in vitro. Our aim was to determine the effect of recombinant human FSH (rhFSH) glycosylation variants on inhibin A and B production by rat GCs. Preparative isoelectro focusing was used to isolate more acidic/sialylated (pH <4.00) and less acidic/sialylated (pH >5.00) rhFSH charge analogues. Concanavalin A was used to isolate unbound and firmly bound rhFSH glycoforms on the basis of their oligosaccharide complexity. GCs, obtained from oestrogen-primed immature rats, were cultured with either native rhFSH or its glycosylation variants. Inhibin A and B were determined using specific ELISAs. Results were expressed as mean±s.e.m. Under basal conditions, inhibin A was the predominant dimer produced (inhibin A: 673±55; inhibin B: 80±4 pg/ml). More acidic/sialylated charge analogues stimulated inhibin B production when compared to inhibin A at all doses studied; by contrast, less acidic/sialylated charge analogues stimulated inhibin A production and elicited no effect on inhibin B. Glycoforms bearing complex oligosaccharides showed a potent stimulatory effect on inhibin B when compared to inhibin A production (i.e. dose 1 ng/ml: 4.9±0.5 vs 0.9±0.1-fold stimulation, P<0.001). Glycoforms bearing hybrid-type oligosaccharides favoured inhibin A production (i.e. dose 4 ng/ml 2.9±0.1 vs 1.6±0.1-fold stimulation, P<0.05). These results show that the sialylation degree as well as the complexity of oligosaccharides present in the rhFSH molecule may be considered additional factors that differentially regulate dimeric inhibin production by rat GCs.

Stability of the two wings of the coiled-coil domain of ClpB chaperone is critical for its disaggregation activity

The ClpB chaperone forms a hexamer ring and rescues aggregated proteins in co-operation with the DnaK system. Each subunit of ClpB has two nucleotide-binding modules, AAA (ATPase associated with various cellular activities)-1 and AAA-2, and an 85-Å (1 Å=0.1 nm)-long coiled-coil. The coiled-coil consists of two halves: wing-1, leaning toward AAA-1, and wing-2, leaning away from all the domains. The coiled-coil is stabilized by leucine zipper-like interactions between leucine and isoleucine residues of two amphipathic α-helices that twist around each other to form each wing. To destabilize the two wings, we developed a series of mutants by replacing these residues with alanine. As the number of replaced residues increased, the chaperone activity was lost and the hexamer became unstable. The mutants, which had a stable hexameric structure but lost the chaperone activities, were able to exert the threading of soluble denatured proteins through their central pore. The destabilization of wing-1, but not wing-2, resulted in a several-fold stimulation of ATPase activity. These results indicate that stability of both wings of the coiled-coil is critical for full functioning of ClpB, but not for the central-pore threading of substrate proteins, and that wing-1 is involved in the communication between AAA-1 and AAA-2.

Swelling-activated transport of taurine in cultured gill cells of sea bass: physiological adaptation and pavement cell plasticity

We have investigated volume-activated taurine transport and ultrastructural swelling response of sea bass gill cells in culture, assuming that euryhaline fish may have developed particularly efficient mechanisms of salinity adaptation. In vivo, when sea basses were progressively transferred from seawater to freshwater, we noticed a decrease in blood osmotic pressure. When gill cells in culture were subjected to 30% hypotonic shock, we observed a five-fold stimulation of [3H]taurine efflux. This transport was reduced by various anion channel inhibitors with the following efficiency: 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) > niflumic acid > DIDS = diphenylamine-2-carboxylic acid. With polarized gill cells in culture, the hypotonic shock produced a five-fold stimulation of apical taurine transport, whereas basolateral exit was 25 times higher. Experiments using ionomycin, thapsigargin, BAPTA-AM, or removal of extracellular calcium suggested that taurine transport was regulated by external calcium. The inhibitory effects of lanthanum and streptomycin support Ca2+ entry through mechanosensitive Ca2+ channels. Branchial cells also showed hypotonically activated anionic currents sensitive to DIDS and NPPB. Similar pharmacology and time course suggested the potential existence of a common pathway for osmosensitive taurine and Cl− efflux through volume-sensitive organic osmolyte and anion channels. A three-dimensional structure study revealed that respiratory gill cells began to swell only 15 s after hypoosmotic shock. Apical microridges showed membrane outfoldings: the cell surface became smoother with a progressive disappearance of ridges. Therefore, osmotic swelling may not actually induce membrane stretch per se, inasmuch as the microridges may provide a reserve of surface area. This work demonstrates mechanisms of functional and morphological plasticity of branchial cells during osmotic stress.

The α-subunit of AMPK is essential for submaximal contraction-mediated glucose transport in skeletal muscle in vitro

AMP-activated protein kinase (AMPK) is a key signaling protein in the regulation of skeletal muscle glucose uptake, but its role in mediating contraction-induced glucose transport is still debated. The effect of contraction on glucose transport is impaired in EDL muscle of transgenic mice expressing a kinase-dead, dominant negative form of the AMPKα2 subunit (KD-AMPKα2 mice). However, maximal force production is reduced in this muscle, raising the possibility that the defect in glucose transport was due to a secondary decrease in force production and not impaired AMPKα2 activity. Generation of force-frequency curves revealed that muscle force production is matched between wild-type (WT) and KD-AMPKα2 mice at frequencies ≤50 Hz. Moreover, AMPK activation is already maximal at 50 Hz in muscles of WT mice. When EDL muscles from WT mice were stimulated at a frequency of 50 Hz for 2 min (200-ms train, 1/s, 30 volts), contraction caused an ∼3.5-fold activation of AMPKα2 activity and an ∼2-fold stimulation of glucose uptake. Conversely, whereas force production was similar in EDL of KD-AMPKα2 animals, no effect of contraction was observed on AMPKα2 activity, and glucose uptake stimulation was reduced by 50% ( P < 0.01) As expected, 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranosyl 5′-monophosphate (AICAR) caused a 2.3-fold stimulation of AMPKα2 activity and a 1.7-fold increase in glucose uptake in EDL from WT mice, whereas no effect was detected in muscle from KD-AMPKα2 mice. These data demonstrate that AMPK activation is essential for both AICAR and submaximal contraction-induced glucose transport in skeletal muscle but that AMPK-independent mechanisms are also involved.

Imatinib Mesylate Induced Reversal of Leukemic Gene Phenotype in HL60 Cells Coincides with Stimulation of Oxidative Metabolism.

Besides blockade of tyrosine kinases such as c-kit, Imatinib mesylate (IM) regulates glucose flux through downregulation of GLUT-1 transporters in human leukemia cells. This mechanism has the potential to induce regression of type 2 diabetes and hyperlipidemia as observed in patients with chronic myeloid leukemia or hypereosinophilic syndrome. In addition, there is a stimulatory effect of IM on differentiation of human mesenchymal stem cells. Its synergism with retinoic acid or low dose Ara-C is applied in treatment of acute myeloid leukemia (AML). Thus, the AML-derived c-kit positive cell line HL60 was chosen for studying the effect of IM on expression of genes associated with differentiation and metabolism. We analysed the possible feedback on transcription factors (AML1 and AML3) and consequences regarding differentiation and metabolism - associated genes. Quantitative reverse transcriptase PCR analyses revealed that IM treatment of HL60 cells downregulates mRNA synthesis of AML1 and AML3 by 70% without affecting transcription of the c-abl tyrosine kinase. IM reduces expression of CD34 mRNA from 20% to 6% of the housekeeping gene G6PD. The appearance of differentiated cells was accompanied by a remarkable stimulation of mRNAs from CD11b and CD14 (monocyte markers) reaching 4-fold higher expression levels relative to G6PD. This was associated with an increased proportion of osteocalcin (OCN), which has recently been shown to enhance mitochondrial activity. A 2-fold stimulation of a fat-metabolism associated mitochondrial palmitoyltransferase (CPT1B) and 10-fold stimulation of microsomal carnitine palmitoyltransferase and the carnitine transporter OCTN2 supports previous data indicating an IM-associated stimulation of oxidative metabolism resulting in a regression of type-2 diabetes and hyperlipidemia. Our current investigations show that IM-associated attenuation of cell proliferation inhibited transcription factors AML1 and AML3 and triggered differentiation in the leukemic cell line HL60, as reflected by altered mRNA expression of surface marker genes. The IM - induced stimulation of lipid metabolism in HL60 confirms previous data indicating a reversal of the Warburg effect in K562 cells without cytocidal activity. This indicates a similar mechanism as known for other drugs and strategies targeting glucose or fat metabolism, which are discussed in the context of cancer prevention and treatment.

A Homogeneous Fluorescent Cell-Based Assay for Detection of Heterologously Expressed Nitric Oxide Synthase Activity

Arhodamine-derived, membrane-permeable fluorophore (DAR-4MAM) sensitive to nitric oxide production has been developed recently. The authors evaluated this reagent in both 96 and 384-well formats using heterologously expressed neuronal nitric oxide synthase (nNOS). nNOS transfected into HEK-293T cellswas stimulated by the addition of ionomycin. The calcium mobilization resulting from ionomycin treatment of nNOS-expressing 293T cells induced a robust increase in emission intensity, as measured using a standard rhodamine filter set. The effect was time dependent, and a 3 to 4-fold stimulation could be achieved in a 2-h time period. Ionomycin-dependent nitric oxide (NO) production was completely inhibited by several arginine analogs at micromolar concentrations (e.g., L-NAME IC 50= 3.0 µ M). Several arginine analog inhibitors of nNOS were revealed to be differentially reversible over increasing substrate concentrations. The assay is a facile method for characterizing inhibitors of nNOS in a relatively unperturbed cell environment.