Type 2 diabetes is associated with suppression of autophagy and lipid accumulation in ß-cells.

Both type 2 diabetes (T2D) and obesity are characterized by excessive hyperlipidaemia and subsequent lipid droplet (LD) accumulation in adipose tissue. To investigate whether LDs also accumulate in ß-cells of T2D patients, we assessed the expression of PLIN2, a LD-associated protein, in non-diabetic (ND) and T2D pancreata. We observed an up-regulation of PLIN2 mRNA and protein in ß-cells of T2D patients, along with significant changes in the expression of lipid metabolism, apoptosis and oxidative stress genes. The increased LD buildup in T2D ß-cells was accompanied by inhibition of nuclear translocation of TFEB, a master regulator of autophagy and by down-regulation of lysosomal biomarker LAMP2. To investigate whether LD accumulation and autophagy were influenced by diabetic conditions, we used rat INS-1 cells to model the effects of hyperglycaemia and hyperlipidaemia on autophagy and metabolic gene expression. Consistent with human tissue, both LD formation and PLIN2 expression were enhanced in INS-1 cells under hyperglycaemia, whereas TFEB activation and autophagy gene expression were significantly reduced. Collectively, these results suggest that lipid clearance and overall homeostasis is markedly disrupted in ß-cells under hyperglycaemic conditions and interventions ameliorating lipid clearance could be beneficial in reducing functional impairments in islets caused by glucolipotoxicity.

Docosahexaenoic acid (DHA): a modulator of microglia activity and dendritic spine morphology.

BACKGROUND: Recent studies have revealed that excessive activation of microglia and inflammation-mediated neurotoxicity are implicated in the progression of several neurological disorders. In particular, chronic inflammation in vivo and exposure of cultured brain cells to lipopolysaccharide (LPS) in vitro can adversely change microglial morphology and function. This can have both direct and indirect effects on synaptic structures and functions. The integrity of dendritic spines, the postsynaptic component of excitatory synapses, dictates synaptic efficacy. Interestingly, dysgenesis of dendritic spines has been found in many neurological diseases associated with ω-3 polyunsaturated fatty acid (PUFA) deficiency and cognitive decline. In contrast, supplemented ω-3 PUFAs, such as docosahexaenoic acid (DHA), can partly correct spine defects. Hence, we hypothesize that DHA directly affects synaptic integrity and indirectly through neuron-glia interaction. Strong activation of microglia by LPS is accompanied by marked release of nitric oxide and formation of lipid bodies (LBs), both dynamic biomarkers of inflammation. Here we investigated direct effects of DHA on synaptic integrity and its indirect effects via microglia in the hippocampal CA1 region. METHODS: Microglia (N9) and organotypic hippocampal slice cultures were exposed to the proinflammagen LPS (100 ng/ml) for 24 h. Biochemical and morphological markers of inflammation were investigated in microglia and CA1 regions of hippocampal slices. As biomarkers of hyperactive microglia, mitochondrial function, nitric oxide release and LBs (number, size, LB surface-associated proteins) were assessed. Changes in synaptic transmission of CA1 pyramidal cells were determined following LPS and DHA (25-50 µM) treatments by recording spontaneous AMPA-mediated miniature excitatory postsynaptic currents (mEPSCs). RESULTS: Microglia responded to LPS stimulation with a significant decrease of mitochondrial function, increased nitric oxide production and an increase in the formation of large LBs. LPS treatment led to a significant reduction of dendritic spine densities and an increase in the AMPA-mediated mEPSC inter-event interval (IEI). DHA normalized the LPS-induced abnormalities in both neurons and microglia, as revealed by the restoration of synaptic structures and functions in hippocampal CA1 pyramidal neurons. CONCLUSION: Our findings indicate that DHA can prevent LPS-induced abnormalities (neuroinflammation) by reducing inflammatory biomarkers, thereby normalizing microglia activity and their effect on synaptic function.

Caspase-1 activity in microglia stimulated by pro-inflammagen nanocrystals.

Although caspase-1 is a key participant in inflammation, there is no sensitive assay to measure its enzymatic activity in real time in cells or animals. Here we describe a nanosensor for caspase-1 ratiometric measurements, consisting of a rhodamine-labeled, caspase-1 cleavable peptide linked to quantum dots (QDs). Microglia cells were stimulated by lipopolysaccharide (LPS) and by hybrid nanoparticles LPS-QDs. These stimuli activated caspase-1 in microglia monolayers and in the mouse brain, while a selected caspase inhibitor markedly reduced it. LPS-QDs entered into the lysosomal compartment and led to an enlargement of these cellular organelles in the exposed microglia. Both lysosomal swelling and mitochondrial impairment contributed to caspase-1 activation and to the consequent interleukin-1ß release. The results from these studies highlight how the unique properties of QDs can be used to create versatile biotools in the study of inflammation in real time in vivo.

Dynamics and regulation of lipid droplet formation in lipopolysaccharide (LPS)-stimulated microglia.

Lipid droplets (LDs) are neutral lipid-rich organelles involved in many cellular processes. A well-known example is their accumulation in leukocytes upon activation by pro-inflammatory stimuli such as lipopolysaccharides (LPS) derived from gram-negative bacteria. A role of LDs and LD-associated proteins during inflammation in the brain is unknown, however. We have now studied their dynamics and regulation in microglia, the resident immune cells in the brain. We find that LPS treatment of microglia leads to the accumulation in them of LDs, and enhancement of the size of LDs. This induction of LDs was abolished by triacsin C, an inhibitor of triglyceride biosynthesis. LPS strongly activated c-Jun N-terminal kinase (JNK) and p38 MAPK stress signaling pathways and increased the expression of LD-associated protein perilipin-2 (ADRP) in a time-dependent manner. Immunostaining showed that perilipin-2 in LPS-treated microglia predominantly colocalized with LDs. Inhibitors of p38 α/ß (SB203580) and PI3K/Akt pathway (LY294002), but not that of JNK (SP600125), reduced LPS-induced LD accumulation and eliminated the activating effect of LPS on perilipin-2. In addition, cytosolic phospholipase A(2) (cPLA(2)-α), a key enzyme for arachidonic acid release, colocalized with LPS-induced LDs. These observations suggest that LDs may play an important role in eicosanoid synthesis in activated microglia; they provide a novel insight into the regulation of LDs in inflammatory cells of the brain and point to a potential role of p38 α/ß in LPS-induced LD accumulation. Collectively, our findings imply that LD formation and perilipin-2 induction could be microglial biomarkers of inflammation in the central nervous system.

Dynein light chain Tctex-type 1 modulates orexin signaling through its interaction with orexin 1 receptor.

Orexins (OX-A, OX-B) are neuropeptides involved in the regulation of the sleep-wake cycle, feeding and reward, via activation of orexin receptors 1 and 2 (OX1R, OX2R). The loss of orexin peptides or functional OX2R has been shown to cause the sleep disorder, narcolepsy. Since the regulation of orexin receptors remains largely undefined, we searched for novel protein partners of the intracellular tail of orexin receptors. Using a yeast two-hybrid screening strategy in combination with co-immunoprecipitation experiments, we found interactions between OX1R and the dynein light chains Tctex-type 1 and 3 (Dynlt1, Dynlt3). These interactions were mapped to the C-terminal region of the dynein light chains and to specific residues within the last 10 amino acids of OX1R. Hence, we hypothesized that dynein light chains could regulate orexin signaling. In HEK293 cells expressing OX1R, stimulation with OX-A produced a less sustained extracellular signal-regulated kinases 1/2 (ERK1/2) activation when Dynlt1 was co-expressed, while it was prolonged under reduced Dynlt1 expression. The amount of OX1R located at the plasma membrane as well as the kinetics and extent of OX-A-induced internalization of OX1R (disappearance from membrane) were not altered by Dynlt1. However, Dynlt1 reduced the localization of OX1R in early endosomes following initial internalization. Taken together, these data suggest that Dynlt1 modulates orexin signaling by regulating OX1R, namely its intracellular localization following ligand-induced internalization.

Multivalent niacin nanoconjugates for delivery to cytoplasmic lipid droplets.

We report here the design, synthesis, and properties, of multifunctional niacin nanoconjugates based on dendritic, miktoarm and linear backbone nanocarriers, using "click" chemistry. The conjugates were in this instance used to deliver the therapeutic agent niacin to lipid droplets. The desired combination of niacin, a lipophilic fluorescent dye (BODIPY), and polyethylene glycol (PEG), was achieved by covalently linking the desired agents to the selected carrier. The nanocarriers containing niacin and BODIPY were found almost exclusively within cytoplasmic lipid droplets in the cells used in this study (living hepatocytes and microglia), whereas the trifunctional carrier containing niacin, BODIPY and PEG was partially localized within these organelles but also elsewhere in the cytoplasmic compartment. Spectrofluorometric analyses, confocal microscopy and fluorescence cell sorting revealed different rates and extent of multifunctional conjugate(s) internalization in the two cell types. Even micromolar concentrations of the internalized multifunctional conjugates did not cause significant cell death or mitochondrial functional impairment, suggesting that they are suitable candidate nanostructures for lipid droplet imaging and for targeting drugs to these cellular organelles. These studies provide an efficient and easy way to synthesize multifunctional nanocarriers by click chemistry, applicable to the synthesis of related multifunctional nanostructures and to their use in the targeting of cellular organelles, including lipid droplets.

Lipid droplets: their role in nanoparticle-induced oxidative stress.

Lipid droplets are cytoplasmic organelles found in almost all cells under physiological or pathological conditions. Certain nanoparticles can induce lipid droplet formation under oxidative stress conditions. Small metallic nanoparticles such as cadmium telluride (CdTe) nanoparticles, particularly those with incompletely protected surfaces, induce oxidative stress and may inflict damages to several intracellular organelles. The objective of this study was to assess formation of lipid droplets in cells treated with CdTe nanoparticles and relate their status to cell function (mitochondrial activity and cell viability). Multicolor labeling of cellular organelles (lipid droplets and lysosomes) showed that lipid droplets formed in pheochromocytoma (PC12) cells following nanoparticle or oleic acid treatment. Some lipid droplets were found closely apposed to lysosomes suggesting possible communication between these organelles during severe oxidative stress. Combination of microscopy of living cells with cell viability assays showed that oleic acid-induced lipid droplets not only serve as intracellular lipid storage sites but also play a protective role in starving stressed cells. Results from these studies suggest that oleic acid-induced LD in PC12 cells are dynamic and adaptive organelles, which provide energy to starving cells and facilitate their rescue under starvation and exposure to metallic nanoparticles.

Molecular imaging of lipid peroxyl radicals in living cells with a BODIPY-alpha-tocopherol adduct.

An increasing number of reports discuss the role reactive oxygen species (ROS) have in cellular pathologies and cellular signaling processes. Critical to elucidating the underlying chemical mechanism behind these biological processes is the development of novel sensors and reporters with chemical sensitivity and, more importantly, molecular specificity, enabling the spatial and temporal monitoring of a specific ROS concentration in live cells. Here we report for the first time on the application of BODIPY-alpha-Tocopherol adduct (B-TOH), a novel lipophilic fluorescent antioxidant indicator, toward detection of peroxyl radicals in model lipid membranes and their imaging in the lipid membrane of live cells. Studies conducted in model lipid membranes show a 5-fold fluorescence enhancement upon reaction of liposome-embedded B-TOH with peroxyl radicals. The enhancement is independent of the solution pH and membrane composition. In studies in live cells performed under states of growth factor withdrawal and increased oxidative stress, a significant increase in B-TOH emission was also observed. Exogenous sources of free radicals were utilized herein, namely, N,N'-dimethyl-4,4'-bipyridinium dichloride (also known as methyl viologen or paraquat) and uncoated nonemissive CdTe nanoparticles, a source of Cd(2+). The recorded fluorescence intensity of B-TOH was proportional to the concentration of the dye and to the level of cellular oxidative stress. By employing fluorescent dyes such as Lysotracker and Nile Red, we demonstrate the formation of peroxyl radicals in subcellular locations in rat pheochromocytoma (PC12 cells) and in primary mouse hippocampal neural cells under oxidative stress conditions. Specifically, we observed peroxyl radicals in lysosomes. The assessment of the subcellular distribution of B-TOH in living cells deprived from growth factors and/or under oxidative stress may be useful in the future in determining subcellular sites of lipid peroxidation. In summary, results from this study underscore the potential of B-TOH as a sensitive and specific probe enabling the molecular imaging of peroxyl radicals in the lipid membranes of live cells.

Peculiarities of the anticoagulant properties for the newly synthesized preparations of coumarin-related compounds.

A) OBJECTIVES: Investigations of the four synthetic coumarin-related compounds: N' - allylthiouridine-3-carbamoil coumarin (GSH-16), N'- morpholylthiouridine-3-carbamoil coumarin (GSH-17), N'-O-fluor-benzyl-N'-3-carbamoil piperaside coumarin (GSH-10) and 6-nitroallylamide-3-carboxy coumarin (GSH-84), were done for study of their role in the processes of hemocoagulation. B) DESIGN AND METHODS: Investigations were carried out on 120 white rats (180-200g), which were injected intravenously by 0.5 ml and 1.0 ml of 1 % solution of GSH-17 and decapitated after 10 and 30 min after the injection by use of light chloroform narcosis. Separation of the liver was done by simultaneous washing with cold physiological solution. C) RESULTS: It was shown that the hemostabilization action of GSH-17 is highly dose- and time - dependent, with by the pronounced decrease of rat liver thromboplastic activity after 10 and especially 30 min following intravenous injection. D) CONCLUSION: One of the possible mechanisms for haemostatic effect of the studied preparations, particularly GSH-17, probably can be accepted their effect on metabolism of arachidonic acid by lipoxygenase and cycloxygenase mechanisms [9, 10]. The results of this investigations have not only the academic interest, but they have also a significant importance for definite branches of practical medicine as a very effective blood stabilizing factors.