Fibroblasts from type 1 diabetics exhibit enhanced Ca(2+) mobilization after TNF or fat exposure

Abstract

The effects of cytokine and fatty acid treatment on signal transduction in dermal fibroblasts from type 1 diabetics and matched controls were compared. Chronic exposure to TNF, accentuated Ca(2+) mobilization in response to bradykinin (BK) in cells from both controls and diabetics; responses were three-fold greater in cells from diabetics than in controls. Similarly, with chronic exposure to IL-1β, BK-induced Ca(2+) mobilization was accentuated in cells from type 1 diabetics compared to the controls. Pretreatment with the protein synthesis inhibitor cycloheximide or the protein kinase C inhibitor calphostin C prior to the addition of TNF completely abrogated the TNF-induced increment in peak bradykinin response. Ca(2+) transients induced by depleting endoplasmic reticulum (ER) Ca(2+) with thapsigargin were also greater in TNF treated fibroblasts than in untreated cells, with greater increases in cells from diabetics. Exposing fibroblasts for 48 hours to 2 mM oleate also increased both the peak bradykinin response and the TNF-induced increment in peak response, which were significantly greater in diabetics than controls. These data indicate that cells from diabetic patients acquire elevated ER Ca(2+) stores in response to both cytokines and free fatty acids,and thus exhibit greater sensitivity to environmental inflammatory stimuli and elevated lipids.

Figure 1. Representative traces of the effect of 24 hours of exposure to TNF (0.6 nM) or IL-1β (60 fM) on BK-induced Ca²⁺ mobilization.

A. Fura loaded fibroblasts from control and type 1 diabetic donors, previously exposed to 0.6B. Fura loaded fibroblasts from one representative control and type 1 diabetic donor, previously exposed to 60 fM Il-1β for 24 hrs, were stimulated with 1 µM BK at 200 seconds. (Similar traces were obtained for each control, relative, and T1D sample and the summarized data is shown in Figs. 4 and 8).

Figure 2. Time course (A) and concentration dependence (B) of effect of TNF pre-treatment on peak BK responses.

A. Fibroblasts from 3 different donors were treated with TNF (0.6 nM) for 1 to 48 hours. Cells were loaded with fura as described in methods and tested with BK. Each bar represents the mean ± SEM of between 2 and 6 separate determinations. B. A single experiment performed in triplicate with fibroblasts from one control and one diabetic subject.

Figure 3. Summary of incremental effect of BK on Ca²⁺ mobilization in control (A) and T1D (B) fibroblasts.

The change in peak response to BK before and after TNF treatment was measured in fibroblasts from 7 control (A) and 10 diabetic (B) donors (3–8 separate experiments per donor). Each point represents the mean ± SEM of 14 to 34 separate determinations. *Indicates that control is significantly different from diabetic (ANOVA p<0.005).

Figure 4. Comparison of TNF (0.6 nM for 24 hours) effects on BK-induced Ca²⁺ mobilization in type 1 diabetics, their siblings of type 1 diabetics, and control human fibroblasts.

The increment in peak response to BK following TNF treatment was determined in 7 control donors, 3 non-diabetic siblings of type 1 diabetics, and 10 diabetic donors (3–8 separate experiments per donor). Each bar represents the mean of 6 to 18 separate determinations. The boxes across each donor group represent the mean ± SEM for each group of donors. Control, diabetic and sibling groups were significantly different from each other (ANOVA p<0.001).

Figure 5. Representative traces to determine the source of TNF-induced increases in Ca²⁺ in response to BK.

A. Untreated and TNF-treated (0.6 nM for 24 hours) fibroblasts were exposed to 200 nM BK. Addition of 2 mM EGTA 10 seconds prior to BK stimulation did not affect the magnitude of the peak in either untreated or TNF-treated cells, but did eliminate the increase in final Ca²⁺ equilibrium. These are representative traces from one diabetic donor. B. Fura loaded fibroblasts (untreated and TNF treated) from one control and one type 1 diabetic donor were treated with 30 nM thapsigargin at 170 seconds to release Ca²⁺ from the endoplasmic reticulum stores. These experiments were repeated three times with similar results.

Figure 6. Representative trace illustrating the Ca²⁺ set point in permeabilized fibroblasts.

Untreated and TNF-treated (0.6 nM for 24 hours) fibroblasts were suspended in an intracellular buffer containing 1 µM fura-2 free acid, and permeabilized with saponin (60 µg/ml) at 100 seconds. The cells took up Ca²⁺ from the buffer until steady state was reached. These experiments were repeated three times with similar results.

Figure 7. Representative traces of the effect of oleate and oleate plus TNF on peak BK response in fibroblasts from a single type 1 diabetic donor.

These are representative traces of BK responses in fibroblasts from a single diabetic donor. Where indicated, 2 mM oleic acid was added 24 hours before, and throughout the subsequent 24 hour incubation. Cells in basal 5.6 mM glucose were treated with TNF, loaded with fura, and tested with BK as described in Figure 1.

Figure 8. The effect of oleate and TNF on peak BK response in human fibroblasts.

Fibroblasts from 3 relatively unresponsive controls and 4 diabetic donors were used. Where indicated, 2/or TNF was added 24 hours before, and throughout the subsequent 24 hour incubation. Cells were then loaded with fura and tested with BK as described in Figure 1. Data are expressed here as a percentage of the untreated condition (5.6 mM glucose). A. Both TNF and oleic acid had significant effects on peak BK response in diabetic cells (ANOVA p<0.05 and p<0.001 respectively). B. Oleic acid had a significant effect on the sustained steady state Ca²⁺ (p<0.001) in diabetic fibroblasts (p<0.001).

Figure 9. Model of TNF and oleate induced alteration in Ca²⁺ handling.

TNF increases cytosol LC-CoA by inhibting its oxidation. Oleate increases LC-CoA via its cytosolic activation to oleoyl CoA. LC-CoA directly stimulates the ER Ca²⁺-ATPase to increase Ca²⁺ stores and activates PKC leading to an enhanced BK-induced signaling response.