Concomitant administration of SM accentuates adipogenic potential of HA <i>in vitro</i>.

<p><b>A.</b> HO activity, <b>B.</b> Heme concentrations, <b>C.</b> Quantification of Oil Red staining and <b>D.</b> Representative light microscopic (x100) image of Oil Red uptake in differentiated adipocytes following 10 days treatment of 3T3 cells with an adipogenic differentiation cocktail alone or with the addition of protoporphyrins. B, C: *P<0.05, **P<0.01, ***P<0.001 v PBS; ^†P<0.05, ^††P<0.01 v HA. <b>E.</b> Time course changes in gene expression of adipogenic genes including PPAR-γ, Adipoq, FASN in 3T3-L1 cells treated with an adipogenic differentiation cocktail alone or with the addition of 20 µM HA, 20 µM SM or HA+SM. ^†††P<0.001: HA v PBS; ^‡P<0.05,^‡‡‡P<0.001: SM v PBS, ***P<0.001: HA+SM v PBS; ^#P<0.05, ^##P<0.01: HA+SM v HA. P-values derived by 2-way ANOVA with Bonferroni’s multiple comparison post-test. The data are mean ± s.e.m from n = 4/group. F. Representative (200x) images of Oil Red staining of stromal vascular fraction cells from epididymal fat pads of HO-1^+/+ (left) and HO-1^+/− (right) mice either undifferentiated or following differentiation by an adipogenic cocktail in the presence of HA.</p

HA+SM increases islet area but reduces islet macrophage infiltration and expression of iNOS protein.

<p><b>A.</b> Representative images of the islets from each treatment group. β-cells are visualised by staining with anti-insulin antibody (green), with either F4/80⁺ macrophages (upper panel) or iNOS positive cells (lower panel) stained in brown. Scale bars represent 50 µM. Quantification of <b>B.</b> islet cross-sectional surface area; and number of <b>C.</b> F4/80⁺ macrophages or <b>D.</b> iNOS⁺ cells per unit islet area in each treatment group. ^‡P<0.05,^‡‡‡P<0.001 v lean PBS, *P<0.05, **P<0.01, ***P<0.001 v db/db PBS, ^††P<0.01 v db/db HA. P-values derived from Bonferroni’s multiple comparison post-test when P<0.05 by one-way analysis of variance. The data are mean ± s.e.m from n>25 islets/mouse.</p

Clinical parameters in the pilot study examining the efficacy of heme arginate (HA) and L-arginine (LA) in the db/db mouse model of type 2 diabetes.

<p>Clinical parameters in the pilot study examining the efficacy of heme arginate (HA) and L-arginine (LA) in the db/db mouse model of type 2 diabetes.</p

Administration of SM further augmented HO-1 protein expression but abrogated the increase in HO activity induced by HA.

<p>Representative immuno-blots of HO-1 expression in <b>A.</b> Epididymal fat and <b>B.</b> Liver. <b>C.</b> Hepatic HO activity in each treatment group. ***P<0.001, P-values derived from Bonferroni’s multiple comparison post-test where P<0.05 by one-way analysis of variance. Data given are means ± s.e.m.</p

HA±SM improved glycaemic control and β-cell function despite a significant increase in body weight in db/db mice.

<p><b>A.</b> Fasting glucose, <b>B.</b> Body weight, <b>C.</b> HOMA–IR as measure of insulin resistance and <b>D.</b> HOMA-β as measure β-cell function during administration of PBS (n = 4), HA (n = 4), SM (n = 5) and HA+SM (n = 8) to db/db mice for 8 weeks. ^†P<0.05: HA v PBS; *P<0.05, **P<0.01, ***P<0.001: HA+SM v PBS, ^#P<0.05, ^##P<0.01: HA+SM v HA. P-values derived by 2-way ANOVA with Bonferroni’s multiple comparison post-test. Data given are means ± s.e.m.</p

HA±SM reduces cytokine mix (CM) induced pro-inflammatory response in MIN6 β-cell line.

<p>Relative gene expression of <b>A.</b> iNOS, <b>B.</b> IL-1β, C. MCP-1α, <b>D.</b> Cxcl-1 in MIN6 β-cells pre-treated with either PBS, HA, SM or HA+SM prior to stimulation with cytokine mix. <b>E.</b> HO activity in MIN6 β-cells. *P<0.05, **P<0.01, ***P<0.001. P-values derived from Bonferroni’s multiple comparison post-test when P<0.05 by one-way analysis of variance. The data are means ± s.e.m from n = 4 independent experiments/group.</p

Treg depletion in the FoxP3.LuciDTR mouse does not increase susceptibility to IRI.

<p>FoxP3.LuciDTR mice or wildtype control (n = 5–10 per group) received 25 ng/g DT 24 hours prior to ischemic insult. ALT release [a] and histological injury score [b] did not differ between groups. DT treatment of DTR animals effected almost total depletion of Treg from the circulation [c,d], spleen [e] and liver [f].</p

Adoptive transfer of pre-activated iTreg does not protect animals from IRI.

<p>iTreg were generated <i>in vitro</i> from sorted CD62L high, FoxP3- naïve T cells cultured in the presence of TGFβ and IL-2 for 5 days before flow sorting to maximize purity [a-d]. Successful transfer was confirmed by detection of FoxP3 GFP+cells in spleen [e] and liver [f]. No difference was detected in injury severity between iTreg supplemented and PBS treated control animals at 3 or 24 hours of reperfusion (n = 4–5 per group) [g].</p

Adoptive transfer of pre-activated nTreg does not protect animals from IRI.

<p>nTreg were obtained from FoxP3 GFP mouse spleens by flow sorting followed by expansion <i>in vitro</i> for 21 days and further flow-sorting for purity [a–c]. Flow cytometry of spleen and liver confirmed successful transfer [d,e]. There was no difference in injury severity between nTreg supplemented and control animals at 3 hours of reperfusion (n = 4 per group) [f].</p

Treg mobilization during reperfusion is not enhanced by IPC.

<p>Mice (n = 5–8 per group) were subjected to ischemia reperfusion injury with or without ischemic preconditioning and killed at 3 or 24 hours. Ischemic preconditioning protected the liver from injury both in terms of ALT release [a] and histological injury score [b] measured at 24 hours. CD4+FoxP3+cells were mobilized into the circulation during reperfusion [c]. Hepatic CD4+FoxP3+cells increased over the same time period [d]. Total CD3+lymphocytes were stable throughout reperfusion [e]. FlowCytomix was used to profile circulating chemo/cytokines. Rises were detected in CXCL-10/IP-10 [f], CXCL-1/KC [g], IL-6 [h] and GMCSF [i]. Other analytes (IL-1α, IL-1β, IL-2, IFNγ, IL-17 and IL-17F, and the Treg cytokine IL-10) were not detected.</p