Competing risk analysis of site-specific cancer mortality for glargine exposure.

a<p>Although not shown here, the model also included age at first insulin prescription (FIP), gender, time between screening and FIP, treatment intensity level and all other available oral or insulin categories as both cumulative exposure and time-dependent ever exposed terms (see Methods).</p>b<p>SHR, subhazard ratios, similar to hazard ratios from the classic Cox regression.</p>c<p>Significant result rounded up to <0.05 due to very low estimated SHR.</p

Cancer deaths cumulative incidence functions.

<p>Green line, females exposed to glargine; brown line, females unexposed to glargine; blue lines, males exposed to glargine; red line, males unexposed to glargine.</p

Characteristics of ever and never glargine users.

a<p>Percent within ever and never users, respectively.</p><p>OGLD oral glucose lowering drugs,TIL treatment intensity level (see Methods), FIP first insulin prescription.</p>NS<p>not significant, *p<0.05, **p<0.01, ***p<0.001 vs. ever glargine users.</p

Effect of Sfrp5 on Cytokine Release and Insulin Action in Primary Human Adipocytes and Skeletal Muscle Cells

<div><p>Secreted frizzled-related protein 5 (Sfrp5) is an adipokine with anti-inflammatory and insulin-sensitizing properties in mice. However, the mechanism of Sfrp5 action, especially in humans, is largely unknown. Therefore, cytokine release and insulin signaling were analyzed to investigate the impact of Sfrp5 on inflammation and insulin signaling in primary human adipocytes and skeletal muscle cells (hSkMC). Sfrp5 neither affected interleukin (IL)-6, monocyte chemoattractant protein-1 (MCP-1) and adiponectin release from human adipocytes, nor IL-6 and IL-8 release from hSkMC. In tumor necrosis factor (TNF) α-treated adipocytes, Sfrp5 reduced IL-6 release by 49% (p<0.05), but did not affect MCP-1 and adiponectin release. In MCP-1-treated hSkMC, Sfrp5 did not affect cytokine secretion. In untreated adipocytes, Sfrp5 decreased the insulin-mediated phosphorylation of Akt-Ser473, Akt-Thr308, GSK3α-Ser21 and PRAS40-Thr246 by 34% (p<0.01), 31% (p<0.05), 37% (p<0.05) and 34% (p<0.01), respectively, and the stimulation of glucose uptake by 25% (p<0.05). Incubation with TNFα increased the phosphorylation of JNK and NFκB, and impaired insulin signaling. When Sfrp5 and TNFα were combined, there was no additional effect on insulin signaling and JNK phosphorylation, but phosphorylation of NFκB was reversed to basal levels. Sfrp5 had no effect on insulin signaling in untreated or in MCP-1 treated hSkMC. Thus, Sfrp5 lowered IL-6 release and NFκB phosphorylation in cytokine-treated human adipocytes, but not under normal conditions, and decreased insulin signaling in untreated human adipocytes. Sfrp5 did not act on hSkMC. Therefore, the cellular actions of Sfrp5 seem to depend on the type of tissue as well as its inflammatory and metabolic state.</p></div

Effect of Sfrp5 on glucose uptake in primary human adipocytes.

<p>Mature adipocytes were kept untreated, exposed to 100/ml Sfrp5 for 24 h, when indicated (+) stimulated with insulin (30 min; 100 nM), whereafter glucose uptake was determined. The incorporated amount of 2-deoxy-D-[1-³H]glucose was normalized for protein content and expressed as mean ± standard error of the mean of four independent experiments using cells from different donors. The values obtained for Sfrp5-untreated, insulin-treated cells were considered as control and set at 100%. Differences among groups were calculated by two-way ANOVA followed by Bonferroni multiple comparison analysis. #, indicates <i>p</i><0.05 for the effect of insulin stimulation; *, <i>p</i><0.05 for the effect of Sfrp5.</p

Effect of Sfrp5 on adipokine and myokine release from cytokine-stimulated cells.

<p>Primary human adipocytes (A–C) and primary human skeletal muscle cells (D–E) were exposed to Sfrp5 (4 h; 100 ng/ml) prior to incubation with TNFα (24 h; 5 nmol/l) or MCP-1 (18 h; 2 ng/ml). Cytokine release by the adipocytes and myotubes was quantified by ELISA, and expressed as mean ± standard error of the mean (A–C: n = 5; D–E: n = 4). Differences among the various conditions were analyzed by Friedman's test followed by Dunn's multiple comparison test; ### indicates <i>P</i><0.001; ##, <i>P</i><0.01 versus cells kept untreated (basal); *, <i>P</i><0.05 for the effect of the additional Sfrp5 incubation versus TNFα alone.</p

Effect of Sfrp5 on insulin signaling in primary human adipocytes.

<p>Primary human adipocytes were kept untreated, exposed to Sfrp5 for 24/ml TNFα for 24 h with or without a 4 h preincubation with Sfrp5. Then, when indicated (+) cells were stimulated with insulin (10 min; 100 nM). Cell lysates were analyzed for phosphorylation of Akt-Thr308 (A), Akt-Ser473 (B), GSK3α-Ser21 (C) and PRAS40-Thr246 (D) by Western blotting. Phosphorylation signals were normalized for GAPDH protein abundance and expressed as mean ± standard error of the mean of five independent experiments using cells from different donors. The values obtained for Sfrp5-untreated, insulin-treated cells were considered as control and set at 100%. Differences among groups were calculated by two-way ANOVA followed by Bonferroni multiple comparison analysis. #, indicates <i>p</i><0.05 for the effect of insulin stimulation; ***, <i>p</i><0.001; **, <i>p</i><0.01; *, <i>p</i><0.05 versus untreated cells.</p

Effect of Sfrp5 on adipokine and myokine release (unstimulated conditions).

<p>Primary human adipocytes (A–C) and primary human skeletal muscle cells (D–E) were exposed to increasing amounts of Sfrp5 for 24 h. Cytokine release by the adipocytes and myotubes was quantified by ELISA, and expressed as mean ± standard error of the mean (A–C: n = 5; D–E: n = 4).</p

Perturbation of the molecular clockwork in the SCN of non-obese diabetic mice prior to diabetes onset

<p>Circadian disruption is associated with the development of diabetes. Non-obese diabetic (NOD) mice show abnormal diurnal profiles in energy balance and locomotor activity suggesting circadian misalignment. Therefore, we analyzed cFos and mPER1 as markers for rhythmic neuronal activity within the suprachiasmatic nucleus (SCN) of wildtype (WT) and non-diabetic (nNOD) as well as acutely diabetic NOD (dNOD) mice. cFos levels show a day/night difference in both WT and nNOD but not in dNOD. mPER1 levels did not show a day/night difference in both nNOD and dNOD. This suggests that disruption of SCN rhythmicity in NOD mice precedes the actual onset of diabetes.</p

Effect of Sfrp5 and TNFα on inflammation signaling in primary human adipocytes.

<p>Primary human adipocytes were kept untreated, exposed to Sfrp5 for 24/ml TNFα for 24 h with or without a 4 h preincubation with Sfrp5. Cell lysates were analyzed for phosphorylation of NFκB-Ser536 (A) and JNK-Tyr183/Thr185 (B) by Western blotting. Phosphorylation signals were normalized for GAPDH protein abundance and expressed as mean ± standard error of the mean of seven independent experiments using cells from different donors. The values obtained for untreated cells were considered as control and set at 100%. Differences among groups were calculated by ANOVA followed by Bonferroni multiple comparison analysis. ***, <i>p</i><0.001 versus untreated cells; ††, <i>p</i><0.01 for the effect of Sfrp5 versus TNFα.</p