The SNARE Protein SNAP23 and the SNARE-Interacting Protein Munc18c in Human Skeletal Muscle Are Implicated in Insulin Resistance/Type 2 Diabetes

OBJECTIVE-Our previous studies suggest that the SNARE protein synaptosomal-associated protein of 23 kDa (SNAP23) is involved in the link between increased lipid levels and insulin resistance in cardiomyocytes. The objective was to determine whether SNAP23 may also be involved in the known association between lipid accumulation in skeletal muscle and insulin resistance/type 2 diabetes in humans, as well as to identify a potential regulator of SNAP23. RESEARCH DESIGN AND METHODS-We analyzed skeletal muscle biopsies from patients with type 2 diabetes and healthy, insulin-sensitive control subjects for expression (mRNA and protein) and intracellular localization (subcellular fractionation and immunohistochemistry) of SNAP23, and for expression of proteins known to interact with SNARE proteins. Insulin resistance was determined by a euglycemic hyperinsulinemic clamp Potential mechanisms for regulation of SNAP23 were also investigated in the skeletal muscle cell line L6. RESULTS-We showed increased SNAP23 levels in skeletal muscle from patients with type 2 diabetes compared with that from lean control subjects Moreover, SNAP23 was redistributed from the plasma membrane to the microsomal/cytosolic compartment in the patients with the type 2 diabetes Expression of the SNARE-interacting protein Munc18c was higher in skeletal muscle from patients with type 2 diabetes Studies in L6 cells showed that Munc18c promoted the expression of SNAP23. CONCLUSIONS-We have translated our previous in vitro results into humans by showing that there is a change in the distribution of SNAP23 to the interior of the cell in skeletal muscle from patients with type 2 diabetes. We also showed that Munc18c is a potential regulator of SNAP23. Diabetes 59: 1870-1878, 201

Anti-infectious and anti-inflammatory activities of lactoferrin and fragments thereof

Lactoferrin (LF), a major protein present in milk, mucosal secretions and secondary granules of neutrophils, has been suggested to participate in host defense at mucosal surfaces and to mediate anti-inflammatory activities. A pepsin-derived fragment of LF has been shown to contain the antibacterial domain of the protein. Despite this proposed important dual capacity of LF at the mucosal membranes there are few in vivo studies to support these effects. Our aim was to investigate if anti-infectious or anti-inflammatory activities on mucosal surfaces could be mediated by perorally given LF or synthetic fragments of the antibacterial region of LF in experimental animal models, to gain insight into how LF mediates the anti-inflammatory activities in vitro, and to define the sequence in the antibacterial region of the LF molecule responsible for the antimicrobial activity. Experimental mouse models of urinary tract infection (UTI) induced by E.coli O6K5 and dextran sulphate (DX)-induced acute colitis were used. The number of E.coli present in the urinary tract and the urinary and systemic inflammatory response (urinary leukocytes, urinary and systemic IL-6 levels) in mice with UTI were reduced by the LF treatment compared to the control group. Mice treated with LF peptide fragments (HLD1 and 2) also showed reduced numbers of bacteria in the kidneys. The perorally given LF was found to pass over to the circulation and urine. HLD2 mediated significant bactericidal activity against E.coli when tested in vitro in mouse urine. The damaging effects induced by DX exposure (presence of blood in the faeces, colon shortening, IL-1b serum levels, crypt score) were delayed or reduced in mice treated with LF or the peptides. The number of inflammatory cells present in the colon after 7 days of DX exposure (F4/80 macrophages, CD4- and TNF-a- positive cells) was lower in the LF treated group compared to the control. LF also reduced shortening of the colon when given orally to animals with an already established inflammatory response as induced by two days of DX exposure. LF was shown to down-regulate the secretion of TNF-a, IL-1b, IL-6, IL-8 and IL-10 in monocytic cell lines (THP-1, Mono Mac 6) stimulated with LPS. The down regulation of the cytokines was reflected at the transcriptional level. Thus LPS-induced TNF-a-, IL-1b-, IL-6-, and IL-8 mRNA as shown by reversed transcription PCR were reduced as well. The known binding of LF to LPS could not explain the reduced cytokine mRNA expression and protein secretion since the effects were observed also when LF was added 30 min after the LPS to the cell assay. In addition, also IL-1b induced IL-6 secretion was down-regulated in the presence of LF. Moreover, LF was detected by immunocytochemistry in the cell nucleoli already after 30 min of incubation with the THP-1 cells and found by electromobility shift assay to decrease the binding of nuclear factor (NF)-kB to the TNF-a promoter. The antimicrobial activity against E.coli, S.aureus and C.albicans of synthetic peptides homologous to the surface exposed a-helix and b-sheet region from the N-terminal end of human LF showed that a short region comprising 12-15 a.a. corresponding to the major part of the helix region were optimal for the killing activity against all three microorganisms. In addition certain amino acids such as cystein, and hydrophobic and positively charged amino acids in the 12-amino acid long peptide were found to be important for the expression of antimicrobial activity. In summary, orally given LF can reduce infection and inflammation in a remote site such as the urinary tract, and mediates anti-inflammatory activities in the colon. This dual effect may partly reside in the antimicrobial region of the LF molecule, since synthetic fragments also provide similar activities. One possibly important mechanism of its anti-inflammatory effects is through the ability to down-regulate cytokine production via interference with the transcription factor NF-kB. The anti-infectious and anti-inflammatory activities of LF on mucosal surfaces is being utilized by the suckling child which obtains large amounts of LF via the maternal milk. However, therapeutic use of LF or its fragments may be possible in other fields of application

Glucosylceramide modifies the LPS-induced inflammatory response in macrophages and the orientation of the LPS/TLR4 complex in silico

Toll-like receptor 4 (TLR4) is activated by bacterial lipopolysaccharide (LPS), which drives the production of proinflammatory cytokines. Earlier studies have indicated that cholesterol-and glycosphingolipid-rich subregions of the plasma membrane (lipid domains) are important for TLR4-mediated signaling. We report that inhibition of glucosylceramide (GluCer) synthase, which resulted in decreased concentrations of the glycosphingolipid GluCer in lipid domains, reduced the LPS-induced inflammatory response in both mouse and human macrophages. Atomistic molecular dynamics simulations of the TLR4 dimer complex (with and without LPS in its MD-2 binding pockets) in membranes (in the presence and absence of GluCer) showed that: (1) LPS induced a tilted orientation of TLR4 and increased dimer integrity; (2) GluCer did not affect the integrity of the LPS/TLR4 dimer but reduced the LPS-induced tilt; and (3) GluCer increased electrostatic interactions between the membrane and the TLR4 extracellular domain, which could potentially modulate the tilt. We also showed that GCS inhibition reduced the interaction between TLR4 and the intracellular adaptor protein Mal. We conclude that the GluCer-induced effects on LPS/TLR4 orientation may influence the signaling capabilities of the LPS/TLR4 complex by affecting its interaction with downstream signaling proteins.Peer reviewe

ARF6 regulates neuron differentiation through glucosylceramide synthase.

The small GTPase ADP ribosylation factor 6 (ARF6) mediates endocytosis and has in addition been shown to regulate neuron differentiation. Here we investigated whether ARF6 promotes differentiation of Neuro-2a neuronal cells by modifying the cellular lipid composition. We showed that knockdown of ARF6 by siRNA in Neuro-2a cells increased neuronal outgrowth as expected. ARF6 knockdown also resulted in increased glucosylceramide levels and decreased sphingomyelin levels, but did not affect the levels of ceramide or phospholipids. We speculated that the ARF6 knockdown-induced increase in glucosylceramide was caused by an effect on glucosylceramide synthase and, in agreement, showed that ARF6 knockdown increased the mRNA levels and activity of glucosylceramide synthase. Finally, we showed that incubation of Neuro-2a cells with the glucosylceramide synthase inhibitor D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) normalized the increased neuronal outgrowth induced by ARF6 knockdown. Our results thus show that ARF6 regulates neuronal differentiation through an effect on glucosylceramide synthase and glucosylceramide levels

Lack of RAC1 in macrophages protects against atherosclerosis.

The Rho GTPase RAC1 is an important regulator of cytoskeletal dynamics, but the role of macrophage-specific RAC1 has not been explored during atherogenesis. We analyzed RAC1 expression in human carotid atherosclerotic plaques using immunofluorescence and found higher macrophage RAC1 expression in advanced plaques compared with intermediate human atherosclerotic plaques. We then produced mice with Rac1-deficient macrophages by breeding conditional floxed Rac1 mice (Rac1fl/fl) with mice expressing Cre from the macrophage-specific lysosome M promoter (LC). Atherosclerosis was studied in vivo by infecting Rac1fl/fl and Rac1fl/fl/LC mice with AdPCSK9 (adenoviral vector overexpressing proprotein convertase subtilisin/kexin type 9). Rac1fl/fl/LC macrophages secreted lower levels of IL-6 and TNF-α and exhibited reduced foam cell formation and lipid uptake. The deficiency of Rac1 in macrophages reduced the size of aortic atherosclerotic plaques in AdPCSK9-infected Rac1fl/fl/LC mice. Compare with controls, intima/media ratios, the size of necrotic cores, and numbers of CD68-positive macrophages in atherosclerotic plaques were reduced in Rac1-deficient mice. Moreover, we found that RAC1 interacts with actin-binding filamin A. Macrophages expressed increased RAC1 levels in advanced human atherosclerosis. Genetic inactivation of RAC1 impaired macrophage function and reduced atherosclerosis in mice, suggesting that drugs targeting RAC1 may be useful in the treatment of atherosclerosis

ARF6-dependent neuronal differentiation is normalized after inhibition of glucosylceramide synthase.

<p>(<b>A</b>) Quantification of long outgrowth from Neuro-2a cells transfected with control or ARF6 siRNA in medium with 10% FCS for 48 h and then differentiated for 24 h in medium without serum in the absence or presence of D-PDMP (10 µmol/l). <i>n</i> = 4 per group, ***<i>P</i><0.001 <i>vs</i> control; †††<i>P</i><0.001 <i>vs</i> absence of D-PDMP. (<b>B</b>) Cellular levels of ceramides (Cer), sphingomyelin (SM) and glucosylceramide (GC). <i>n</i> = 4–5 per group, **<i>P</i><0.01 <i>vs</i> control, ***<i>P</i><0.001 <i>vs</i> control.</p

ARF6 knockdown increases glucosylceramide levels and decreases sphingomyelin levels in Neuro-2a cells.

<p>Neuro-2a cells were transfected with control (c) or ARF6 siRNA in medium with 10% FCS and lipids were extracted after 48 h. (<b>A</b>) Cellular levels of ceramides (Cer), sphingomyelin (SM) and glucosylceramide (GC) and (<b>B</b>) phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS) were analyzed as described in the methods section. <i>n</i> = 4 per group, **<i>P</i><0.01, ***<i>P</i><0.001 <i>vs</i> control.</p

ARF6 knockdown increases cellular glucosylceramide levels through increased glucosylceramide synthase activity in Neuro-2a cells.

<p>(<b>A</b>) Schematic overview of the sphingolipid synthesis pathway. (<b>B–D</b>) Neuro-2a cells were transfected with control (c) or ARF6 siRNA in medium with 10% FCS and analyzed after 48 h. (<b>B</b>) mRNA expression of glucosylceramide synthase (GCS) in RNA extracted from Neuro-2a cells (<i>n</i> = 6 per group). (<b>C</b>) Representative image of a TLC plate from a GCS activity assay showing increased levels of synthesized NBD-glucosylceramide in Neuro-2a cells transfected with ARF6 siRNA. (<b>D</b>) Quantification of increased glucosylceramide synthase activity after ARF6 knockdown (<i>n</i> = 4 per group). *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 <i>vs</i> control.</p

Vimentin deficiency in macrophages induces increased oxidative stress and vascular inflammation but attenuates atherosclerosis in mice

Abstract The aim was to clarify the role of vimentin, an intermediate filament protein abundantly expressed in activated macrophages and foam cells, in macrophages during atherogenesis. Global gene expression, lipid uptake, ROS, and inflammation were analyzed in bone-marrow derived macrophages from vimentin-deficient (Vim −/−) and wild-type (Vim +/+) mice. Atherosclerosis was induced in Ldlr −/− mice transplanted with Vim −/− and Vim +/+ bone marrow, and in Vim −/− and Vim +/+ mice injected with a PCSK9 gain-of-function virus. The mice were fed an atherogenic diet for 12–15 weeks. We observed impaired uptake of native LDL but increased uptake of oxLDL in Vim −/− macrophages. FACS analysis revealed increased surface expression of the scavenger receptor CD36 on Vim −/− macrophages. Vim −/− macrophages also displayed increased markers of oxidative stress, activity of the transcription factor NF-κB, secretion of proinflammatory cytokines and GLUT1-mediated glucose uptake. Vim −/− mice displayed decreased atherogenesis despite increased vascular inflammation and increased CD36 expression on macrophages in two mouse models of atherosclerosis. We demonstrate that vimentin has a strong suppressive effect on oxidative stress and that Vim −/− mice display increased vascular inflammation with increased CD36 expression on macrophages despite decreased subendothelial lipid accumulation. Thus, vimentin has a key role in regulating inflammation in macrophages during atherogenesis