Figure 1.
TLR4 expression on cells in islets.
Representative flow cytometic analysis showing percentages of insulin+TLR4+ cells in pancreatic islets of 8 weeks old db/db and db/+ mice, MIN6 and INS1 cells (A-D). The number indicates percentage of cells within the gate. Number of insulin+ cells (E), CD68+F4/80− (F) and CD68+F480+ (G) macrophages expressing TLR4 in pancreatic islets of 8 weeks old db/+ and db/db mice. Three independent experiments were performed with a pool of 20 mice in each group per experiment. Unpaired T-test ***p<0.001; **p<0.01; *p<0.05.
Figure 2.
TLR4 reduces glucose stimulated insulin secretion.
NMRI islets were exposed to increasing concentrations of glucose and insulin was measured (A). Islets were pre-incubated with LPS for 24 h before the glucose-stimulated insulin secretion was measured using a concentration of 15 mM glucose (B). Data are presented as normalized averages +−/SEM from 3 independent experiments where each experimental was performed in triplicates. Statistical significance was evaluated by an unpaired two-sided parametric t-test assuming equal variance performed on normalized data. *; **; *** represent p<0.05; 0.01; 0.001, respectively, vs. insulin secretion at control islets at 15 mM glucose. **** represent p<0.0001 of insulin secretion at 15 mM versus at 3–10 mM glucose and n.s. represent non-significant using one-way ANOVA.
Figure 3.
LPS induced cytokine production in islets.
Islets from db/+, db/db, and MIN6 cells left untreated or treated with the indicated LPS concentrations for 24 h and media. LPS induced dose-response of IL-6 (A). Release of IL-6 (B), MCP-1 (C), KC (D) and RANTES (E) from db/+ and db/db islets treated with 1 µg/ml LPS or media alone. Data shows mean from 20 mice run in triplicate. All data are shown as mean ± S.E.M. Statistical analysis is derived by student’s T-test*p<0.05; p<0.01; p<0.001.
Figure 4.
Mean fluorescence intensity (MFI) (A and B) and total number (C and D) of CD68+F4/80+ and CD68+F4/80− macrophages expressing TLR4 at indicated age in db/+ and db/db mice. One representative experiment out of three is shown. Unpaired T-test ***p<0.001; **p<0.01; *p<0.05.
Figure 5.
Transiently aggravated TLR4 induced cytokine signature in diabetic splenocytes.
Splenocytes from 8, 16 and 24 weeks old mice were treated with LPS and production of IL-6 (A), TNFα (B) and IL-1β (C) was monitored. Data shows one representative experiment with mean ± S.E.M from 4 mice, Statistical evaluation conducted by two-way ANOVA *p<0.05; p<0.01; p<0.001.
Figure 6.
Accelerated acquisition to produce cytokines in diabetic macrophages.
Splenocytes were stimulated with 100/ml LPS in the presence of 10 µg/ml Bref-A for 6 h. Intracellular cytokines expression of IL-6 (A, D), TNFα (B, E) and IL-1β (C, F) was then determined in CD68+F4/80− and CD68+F4/80+ macrophages respectively. One representative experiment out of two with at least 4 animals in each is shown. Statistical evaluation using unpaired T-test ***p<0.001; **p<0.01; *p<0.05.
Figure 7.
TLR4 induces β-cell apoptosis though release of soluble factors from macrophages.
Db/db islets and MIN6 cells were stimulated with LPS or combined IL-1β and TNFα for 24 h and apoptosis was measured as cytosolic DNA-histone complexes (A). Media from 8-weeks (A), 16-weeks (B) or 24-weeks (C) old db+ or db/db TLR4 activated splenocytes were added to MIN6 cells for 24 h. MIN6 cell apoptosis was quantified as caspase3/7 activity. Data is shown as mean ±S.E.M. from three experiments. Statistical analysis by student T-test were *p<0.05; **p<0.01; ***p<0.001.
Figure 8.
Diabetic splenocytes activated through TLR4 induce enhanced β-cell apoptosis.
MIN6 cells were co-cultured with 8-week old db+ (A), 8-week old db/db (B), 24-week old db+ (C) or 24-week old db/db (D) for 24 h in media and then for an additional 24 h in the presence of absence of increasing concentration of LPS. Apoptosis was quantified as caspase3/7 activity. Data is shown as mean ±S.E.M. from three experiments. Statistical analysis by student T-test were *p<0.05; **p<0.01; ***p<0.001.
Figure 9.
Cytokine induction in MIN6 cells, splenocytes, co-culture of MIN6 and splenocytes and transwell systems with MIN6 cells and splenocytes.
(A). Induction of apoptosis in MIN6 cells cultured for 24 h and then for an additional 24 h in presence of LPS (B). Apoptosis was quantified as caspase 3/7 activity. Data is shown as mean +/− SEM from two experiments.
Figure 10.
Hypothesis of TLR4 mechanisms in diabetic islet dys-function.