SGC-CK2-1 Is an Efficient Inducer of Insulin Production and Secretion in Pancreatic β-Cells

The pyrazolopyrimidine based compound SGC-CK2-1 is a potent and highly specific CK2 inhibitor and a new tool to study the biological functions of protein kinase CK2 irrespective from off-target effects. We used this compound in comparison with the well-established CK2 inhibitor CX-4945 to analyze the importance of CK2 for insulin production and secretion from pancreatic β-cells. Both inhibitors affected the proliferation and viability of MIN6 cells only marginally and downregulated the endogenous CK2 activity to a similar level. Furthermore, both inhibitors increased the message for insulin and boosted the secretion of insulin from storage vesicles. Thus, regarding the high specificity of SGC-CK2-1, we can clearly attribute the observed effects to biological functions of protein kinase CK2.

Neuregulin 4 Downregulation Induces Insulin Resistance in 3T3-L1 Adipocytes through Inflammation and Autophagic Degradation of GLUT4 Vesicles

The adipokine Neuregulin 4 (Nrg4) protects against obesity-induced insulin resistance. Here, we analyze how the downregulation of Nrg4 influences insulin action and the underlying mechanisms in adipocytes. Validated shRNA lentiviral vectors were used to generate scramble (Scr) and Nrg4 knockdown (KD) 3T3-L1 adipocytes. Adipogenesis was unaffected in Nrg4 KD adipocytes, but there was a complete impairment of the insulin-induced 2-deoxyglucose uptake, which was likely the result of reduced insulin receptor and Glut4 protein. Downregulation of Nrg4 enhanced the expression of proinflammatory cytokines. Anti-inflammatory agents recovered the insulin receptor, but not Glut4, content. Proteins enriched in Glut4 storage vesicles such as the insulin-responsive aminopeptidase (IRAP) and Syntaxin-6 as well as TBC1D4, a protein involved in the intracellular retention of Glut4 vesicles, also decreased by Nrg4 KD. Insulin failed to reduce autophagy in Nrg4 KD adipocytes, observed by a minor effect on mTOR phosphorylation, at the time that proteins involved in autophagy such as LC3-II, Rab11, and Clathrin were markedly upregulated. The lysosomal activity inhibitor bafilomycin A1 restored Glut4, IRAP, Syntaxin-6, and TBC1D4 content to those found in control adipocytes. Our study reveals that Nrg4 preserves the insulin responsiveness by preventing inflammation and, in turn, benefits the insulin regulation of autophagy.

Characterization of lipoprotein lipase storage vesicles in 3T3-L1 adipocytes

Lipoprotein lipase (LPL) is a secreted triglyceride lipase involved in the clearance of very-low-density lipoproteins and chylomicrons from circulation. LPL is expressed primarily in adipose and muscle tissues and transported to the capillary lumen. LPL secretion is regulated by insulin in adipose tissue, however few studies have examined the regulatory and trafficking steps involved in secretion. Here we describe the intracellular localization and insulin-dependent trafficking of LPL in 3T3-L1 adipocytes. We compared LPL trafficking to the better characterized trafficking pathways taken by leptin and GLUT4. We show that LPL trafficking shares some characteristics of these other pathways, but that LPL subcellular localization and trafficking are distinct from GLUT4 and leptin. LPL secretion occurs slowly in response to insulin and rapidly in response to the calcium ionophore ionomycin. This regulated trafficking is dependent on Golgi protein kinase D and the ADP-ribosylation factor GTPase ARF1 localized to caveolar membrane domains. Together, these data give support to a new trafficking pathway for soluble cargo active in adipocytes.

Identification of Sortilin Alternatively Spliced Variants in 3T3L1 Adipocytes

Type 2 diabetes mellitus (T2DM) is a chronic and progressive metabolic disease with no cure. Adipocytes play a crucial role in glycemic regulation and take up circulating glucose in response to insulin signaling. In T2DM, translocation of major glucose transporter 4 (Glut4) from cytoplasmic locations to the plasma membrane is impaired. Sortilin is an important constituent of Glut4 storage vesicles and interacts with guiding proteins to determine location of Glut4 in the trans-Golgi network. Sortilin levels are shown to affect adipocyte function. Using mouse 3T3L1 adipocytes, we demonstrate that alternative splicing of sortilin pre-mRNA results in an inclusion of an exon (17b) between exons 17 and 18 in the 10CC motif of the VPS10p domain crucial for ligand interaction. Sort17b expression correlates to insulin resistance and over-expression of Sort17b decreases glucose uptake in adipocytes. Using co-immunoprecipitation assays, we demonstrate that Sort17b is a strong binding partner of Glut4. Using bioinformatic analysis, we show that this insertion results in a novel intrinsically disordered region and has potential sites of proteolytic cleavage. Our study is the first to describe sortilin’s alternatively spliced variants in adipocytes and their effects on glucose uptake. As a broader approach, the research demonstrates the impact of a post-transcriptional event on the metabolic fate of adipocytes in conditions of insulin resistance.

Novel and Converging Ways of NOX2 and SOD3 in Trafficking and Redox Signaling in Macrophages

Macrophages and related tissue macrophage populations use the classical NADPH oxidase (NOX2) for the regulated production of superoxide and derived oxidants for pathogen combat and redox signaling. With an emphasis on macrophages, we discuss how sorting into secretory storage vesicles, agonist-responsive membrane trafficking, and segregation into sphingolipid and cholesterol-enriched microdomains (lipid rafts) determine the subcellular distribution and spatial organization of NOX2 and superoxide dismutase-3 (SOD3). We discuss how inflammatory activation of macrophages, in part through small GTPase Rab27A/B regulation of the secretory compartments, mediates the coalescence of these two proteins on the cell surface to deliver a focalized hydrogen peroxide output. In interplay with membrane-embedded oxidant transporters and redox sensitive target proteins, this arrangement allows for the autocrine and paracrine signaling, which govern macrophage activation states and transcriptional programs. By discussing examples of autocrine and paracrine redox signaling, we highlight why formation of spatiotemporal microenvironments where produced superoxide is rapidly converted to hydrogen peroxide and conveyed immediately to reach redox targets in proximal vicinity is required for efficient redox signaling. Finally, we discuss the recent discovery of macrophage-derived exosomes as vehicles of NOX2 holoenzyme export to other cells.

Ligand and Structure-Based Hybrid Screening for Anti-Parkinson Agents and their Pharmacological Evaluation

The behavioral and biochemical antiparkinson effect of 7-hydroxyflavone (7-HF) was evaluated by using virtual screening with an e-pharmacophore and shape-based screening approach, and the compound was screened by using the Sigma Aldrich compound library. Screened hits were filtered based on Lipinski’s rule, absorption, distribution,metabolism,elimination, (software for evaluation) (ADME), and toxicity parameters. The best scoring hit, 7-hydroxy 2 phenyl-4H-chromen-4-one, i.e., 7-HF was selected based on shape similarity (> 0.7), g-score, and conserved interactions. Toxicity assessment of retrieved hits was carried out by Osiris and Lazar programs. This study aims to obtain some potential hits, against various antiparkinson category from reported literature and available online resources, and validate their potency by in vivo, in vitro methods. Reserpine 5 mg/kg produces Parkinson’s like condition by depleting presynaptic catecholamines, particularly dopamine through the process of degranulation of storage vesicles. 7-HF 25, 50, and 100 mg/kg was used as a test compound. Syndopa 275 mg/kg was used as a standard drug. The results demonstrate that treatment with 7-HF improved the total locomotor activity and muscular coordination in the rotarod test. In the open field test, enhanced rearing, grooming duration of mobility, and gripping strength in the chimney test, while a decrease in cataleptic scores in the bar test. 7-HF significantly increases catalase, superoxide dismutase, and reduces glutathione level, while reduced the Malondialdehyde (MDA) level. The total protein concentration was also increased in 7-HF treated groups. The behavioral and biochemical results obtained from this study disclosed a definite neuroprotective role of 7-HF in a dose-dependent manner. It is also clear that 7-HF showed potent and effective antiparkinson activity in a similar way as standard. Interestingly, in behavioral and biochemical studies, 7-HF showed approximately equivalent effects as compared to syndopa.

Recycling resources: silica of diatom frustules as a source for spicule building in Antarctic siliceous demosponges

Dissolved silicon (DSi) is biologically processed to produce siliceous skeletons by a variety of organisms including radiolarians, silicoflagellates, choanoflagellates, plants, diatoms and some animals. In the photic ocean, diatoms are dominant consumers over competing other silicifiers. In Antarctica, where DSi is not particularly limiting, diatoms and sponges coexist in high abundances. Interestingly, diatom ingestion by sponges is a regular feeding strategy there. Although it was known that the diatom organic nutrients are readily metabolized by the sponges, what happened to the inorganic diatom silica skeleton remained unexplored. Here, we have conducted a multi-analytical approach to investigate the processing of diatom silica and whether it is reconverted into sponge silica. We have documented widespread diatom consumption by several demosponges, identifying storage vesicles for the diatom-derived silica by electron microscopy and microanalysis. Diatom-consuming sponges showed upregulation of silicatein and silicase genes, which in addition to the δ 30Si values of their silica, supports that the sponges are converting the ingested diatom silica into sponge silica without much further Si fractionation. Our multidisciplinary approach suggests that the reutilization of diatom silica by sponges is a common feature among Antarctic sponges, which should be further investigated in other latitudes and in other silicifiers.

Zinc dysregulation in slc30a8 (znt8) mutant zebrafish leads to blindness and disrupts bone mineralisation

Zinc is an essential cofactor for many cellular processes including gene transcription, insulin secretion and retinal function. Excessive free Zn2+ is highly toxic and consequently intracellular zinc is tightly controlled by a system of transporters, metallothioneins (MTs) and storage vesicles. Here we describe the developmental consequences of a missense allele of zinc efflux transporter slc30a8 (znt8) in zebrafish. Homozygous slc30a8hu1798 larvae are virtually blind and develop very little or no bone mineral. We show that zinc is stored in pigmented cells (melanophores) of healthy larvae but in slc30a8hu1798 mutants it instead accumulates in the bone and brain. Supporting a role for pigment cells in zinc homeostasis, nacre zebrafish, which lack melanophores, also show disrupted zinc homeostasis. The photoreceptors of slc30a8hu1798 fish are severely depleted while those of nacre fish are enriched with zinc. We propose that developing zebrafish utilise pigmented cells as a zinc storage organ, and that Slc30a8 is required for transport of zinc into these cells and into photoreceptors.

Involvement of PDZ-SAP97 interactions in regulating AQP2 translocation in response to vasopressin in LLC-PK1 cells

Arginine-vasopressin (AVP)-mediated translocation of aquaporin-2 (AQP2) protein-forming water channels from storage vesicles to the membrane of renal collecting ducts is critical for the renal conservation of water. The type-1 PDZ-binding motif (PBM) in AQP2, “GTKA,” is a critical barcode for its translocation, but its precise role and that of its interacting protein partners in this process remain obscure. We determined that synapse-associated protein-97 (SAP97), a membrane-associated guanylate kinase protein involved in establishing epithelial cell polarity, was an avid binding partner to the PBM of AQP2. The role of PBM and SAP97 on AQP2 redistribution in response to AVP was assessed in LLC-PK1 renal collecting cells by confocal microscopy and cell surface biotinylation techniques. These experiments indicated that distribution of AQP2 and SAP97 overlapped in the kidneys and LLC-PK1 cells and that knockdown of SAP97 inhibited the translocation of AQP2 in response to AVP. Binding between AQP2 and SAP97 was mediated by specific interactions between the second PDZ of SAP97 and PBM of AQP2. Mechanistically, inactivation of the PBM of AQP2, global delocalization of PKA, or knockdown of SAP97 inhibited AQP2 translocation as well as AVP- and forskolin-mediated phosphorylation of Ser256 in AQP2, which serves as the major translocation barcode of AQP2. These results suggest that the targeting of PKA to the microdomain of AQP2 via SAP97-AQP2 interactions in association with cross-talk between two barcodes in AQP2, namely, the PBM and phospho-Ser256, plays an important role in the translocation of AQP2 in the kidney.