GLUT1 is up-regulated in SIRT6-KO retina.

<p>a) GLUTl immunoreactivity in cross-section of WT and SIRT6-KO mice retina. Ganglion Cell Layer (GCL), Inner Plexiform Layer (IPL), Inner nuclear Layer (INL) Outer Plexiform Layer (OPL), Outer Nuclear Layer (ONL), Retinal Pigment Epithelium (RPE). GLUT1 protein (b) and mRNA levels (c) were determined by Western blot and RT-PCR respectively. β-actin was used as loading control. Data are mean ± SE (n = 6 eyes/group) **p<0.01</p

SIRT6 is active in the mouse retina.

<p>a) H3K56 acetylation is shown by immunofluoescence. b) Representative Western blot showing protein levels of SIRT6 and the acetylation levels of H3K56 and H3K9 in chromatin preparations from WT and KO mice retinas. Total H3 was used for normalization. c) Quantification of the intensity of bands was determined by using the ImageJ and is represented as arbitrary units. Data are mean ± SE (n = 6 eyes/group). **p<0.01, ***p<0.001</p

Grm6 is down-regulated in SIRT6-KO retinas.

<p>Whole retina mRNA from WT and KO mice was used to profile the expression of several key genes of glutamate receptors involved in the synaptic transmission in an Affymetrix Mouse Gene 2.1 ST DNA microarray. a) Heatmap representing the hierarchical cluster analysis shows the differential expressed mRNAs between WT and SIRT6 KO retinas. The graphic depicts the expression levels of ionotropic AMPA glutamate receptors (Gria1–4), Glutamate receptor, ionotropic kainate (Grik1-2-4-5), Glutamate [NMDA] receptors (Grin1-2a-c) and metabotropic glutamate receptors (Grm1–8). The expression data for the hierarchical clustering image has been row normalized to a range of zero to one with blue representing the row minimum and red representing the row maximum. b) RNA was purified from SIRT6 WT and KO retinas, and Grm6 levels analyzed by RT-PCR. c) immunofluorescence was performed in SIRT6 WT and KO retinas with the indicated antibodies. PKC-alpha was used as a marker for ON bipolar cells. Ganglion Cell Layer (GCL), Inner Plexiform Layer (IPL), Inner nuclear Layer (INL), Outer Plexiform Layer (OPL), Outer Nuclear Layer (ONL), Retinal Pigment Epithelium (RPE). Data are mean ± SE (n = 4) **p<0.01 d) Representative fluorescent images of TUNEL analysis performed in WT and SIRT6 KO retinal sections. Apoptotic nuclei (bright green dots) labeled with fluorescein-dUTP were visualized by fluorescence microscopy. Data are mean ± SE (n  = 3) **p<0.01</p

Retinal functional evaluation.

<p>Representative scotopic (A) and photopic (C) electroretinograms from WT and SIRT6-KO mice at different light intensities (dBs). Plots B and D depict average amplitudes of <i>a</i>-wave and <i>b</i>-wave. Note that the fold decrease of the scotopic <i>a</i>-wave amplitude (8) is greater than the fold decrease of the photopic <i>a</i>-wave amplitude (2,5). Data are mean ± SE (n =  4). **p<0.01, ***p<0.001.</p

Activated T cells undergo massive NAD⁺ depletion upon Nampt inhibition.

<p>A, 3×10⁶ PBLs/well were stimulated (or not, unstim.) with 5 µg/ml PHA, 1 µg/ml con A, or 50 ng/ml PMA and 0.5 µM ionomycin in the presence or absence of the indicated FK866 concentrations. 48 h later, cells were lysed in 0.6 M PCA and NAD⁺ content was measured in neutralized extracts. NAD⁺ levels were normalized to those detected in the absence of FK866. B, Unstimulated or PHA-stimulated PBLs were treated with 33 nM FK866 for 48 h before NAD⁺ content was determined. Absolute NAD⁺ levels are presented. *: p<0.05. C, PBLs were cultured for 48 h with PHA with or without FK866 (33 nM) addition. Subsequently, pyridine dinucleotides levels were measured in acid (NAD⁺ and NAPD⁺) or alkaline (NADH and NADPH) cell extracts. Dinucleotide levels were normalized to those detected without FK866. D, PBLs were incubated with PHA and 33 nM FK866 for the indicated times. Thereafter cells were harvested and NAD⁺ and ATP levels were determined in cell extracts whereas cell viability was assessed by PI-staining and flow cytometry. Results were normalized to the values of FK866-untreated cells. E, Resting or PHA-stimulated PBLs were treated (or not) with 33 nM FK866 in the presence or absence of 1 mM NAD⁺. After five-days viability was assessed determining PI⁻ cells by flow cytometry. Results are means ± SD of five (A) or three (B–E) experiments.</p

Evidence for an involvement of Sirt6 in IFN-γ synthesis.

<p>A, PBLs were cultured for 24 h with or without PHA. Thereafter, Sirt6 levels were detected by Q-PCR. mRNA levels in PHA-stimulated cells were compared to those in unstimulated PBLs. B, C, Jurkat cells were transduced with PRS, (PRS) GFP-sh, or (PRS) S6 sh2, subsequently, Sirt6 mRNA levels or Sirt6 protein levels were determined by Q-PCR (B) and immunoblotting (C). D–F, Jurkat cells transduced with PRS or S6 sh2 were stimulated for 12 h with 5 µg/ml PHA, 50 ng/ml PMA, and 0.5 µM ionomycin. Thereafter, supernatants were harvested and TNF-α (D), IFN-γ (E), and IL-4 (F) levels were determined by ELISA. G, H9 cells transduced with GFP-sh or S6 sh2 were stimulated for 12 h with 5 µg/ml PHA, 50 ng/ml PMA, and 0.5 µM ionomycin. Thereafter, intracellular IFN-γ was detected by intracellular staining. Mean fluorescence intensity for IFN-γ expression is indicated for each histogram. H, 3×10⁶ splenocytes from wild type or <i>Sirt6 KO</i> mice/well were seeded in 24 well plates and stimulated for 24 h with 1 µg/ml Con A. Thereafter, supernatants were harvested and IFN-γ levels were determined by ELISA. *: p<0.05. Results are means ± SD of three experiments (A, B, D–F). Panel C and G are representative of three separate experiments.</p

Nam and Na prevent NAD⁺ shortage and cell death induced by FK866 in human T lymphocytes.

<p>A, PHA-stimulated PBLs were treated (or not) with 33 nM FK866 in the presence or absence of 10 mM Nam or of 10 µM Na. After 48 h, NAD⁺ content was determined (expressed as percentage of NAD⁺ content in FK866-untreated cells). B, PBLs were cultured for 24 h with or without PHA, 1 µg/ml Con A, or 50 ng/ml PMA and 0.5 µM ionomycin. Thereafter, Naprt1 mRNA levels were detected by Q-PCR. mRNA levels in mitogen-stimulated PBLs were compared to those in unstimulated PBLs. C, D, PHA-stimulated PBLs were incubated for five days with or without 10 mM Nam or 10 µM Na in the presence or absence of the indicated FK866 concentrations. Thereafter, cells were imaged by light microscopy (C), and cell viability was determined (D). E, PHA-stimulated PBLs were incubated for five days with or without 33 nM Fk866 in the presence of the indicated concentrations of Nam, Na, or tryptophan (Trp). Viability was subsequently determined. F, PBLs were stimulated with or without PHA in the presence or absence of 1 mM Nam or Na. Thymidine incorporation was measured after 48 h by a 16-h pulse with 0.5 µCi/well [³H]thymidine. D-F each treatment was tested in triplicate wells. Results are means ± SD of three (A, B, F) or four (D, E) experiments. In panel C, one representative experiment out of three is shown.</p

Nampt inhibition with FK866 prevents T lymphocyte proliferation and selectively kills activated T cells.

<p>A, PBLs were seeded in 96-well plates in the presence or absence (unstim.) of PHA and 33 nM FK866. Proliferation was assessed 96 h later by standard [³H]thymidine incorporation assay. B, PBLs were incubated in 96-well plates in the presence or absence of 5 µg/ml PHA, 1 µg/ml Con A, with or without the indicated concentrations of FK866. Five days later viability was detected by PI staining and flow cytometry. Spontanous cell death was 12.2% and 28.1% for PHA- and Con A-stimulated PBLs, respectively. C, D, PBLs were stimulated for 7 days with or without allogeneic mature DCs before FK866 at the indicated concentrations was added. After 5 days viability was assessed by PI staining and flow cytometric analysis using the lymphocyte gate (C). Spontaneous PBL death was 18.4%. D: phenotype of unstimulated or DC-stimulated PBLs. E, Immature or LPS stimulated DCs were cultured for 7 days with 33 nM FK866 before staining with FITC-conjugated Annexin-V and PI and flow cytometry. F, Resting or PHA-stimulated PBLs were treated with 33 nM FK866 for the indicated times and subsequently stained with FITC-conjugated Annexin-V and PI for flow cytometric analysis. Mean values ± SD of five (B) and three (A, C) different donors are presented. D–F One representative experiment out of three is shown.</p

A putative model of Nampt's role in activated T lymphocytes.

<p>Nampt activity is responsible for providing sufficient NAD⁺ supplies during T cell activation. NAD⁺, in turn, is required for ATP synthesis, metabolic reactions, and to replenish NADPH levels. In addition, NAD⁺ represents the substrate of NAD⁺-degrading enzymes such as PARP, CD38, and the sirtuins. Among these, Sirt6 appears to have a central role in IFN-γ and TNF-α production. Nampt inhibitors such as FK866 (and possibly Sirt6 inhibitors) could be used to modulate T cell-mediated immune responses and thereby be beneficial in immune disorders.</p

PARP inhibitors and sirtinol attenuate FK866-induced T cell demise.

<p>A, PBLs were cultured for 24 h with or without PHA. Thereafter, Nampt and PARP1 levels were detected by Q-PCR. mRNA levels in PHA-stimulated cells were compared to those in unstimulated PBLs. B, Resting or PHA-stimulated PBLs were incubated with or without 33 nM FK866 in the presence or absence of 300 µM NU1025, 10 µM PJ34, or 300 µM 3-AB. 48 h later NAD⁺ levels were assessed (presented as % of values in FK866-untreated PBLs). *, p<0.05. C, PHA-stimulated PBLs were incubated for five days with or without 33 nM FK866 in the presence or absence of 300 µM NU1025, 10 µM PJ34, or 300 µM 3-AB. Thereafter, viability was assessed. D, 5×10⁵ Jurkat cells were treated for two days with 500 pM FK866 in the presence or absence of 300 µM NU1025, 5 µM PJ34, or 300 µM 3-AB. Subsequently, NAD⁺ content was determined and expressed as % of values in FK866-untreated Jurkat. E, 3×10⁴ Jurkat cells/well were incubated in 96-well plates with or without 300 pM FK866 in the presence or absence of the indicated concentrations of NU1025, PJ34, or 3-AB. Viability was determined 96 h later by PI cell staining and flow cytometry. F, PBLs were incubated for five days with PHA, with or without 33 nM FK866, in the presence or absence of 30 µM sirtinol. Viability was subsequently assessed by PI staining and flow cytometry. C, E, F, each treatment was tested in triplicate wells. Results are presented as means ± SD of three experiments.</p