Impaired proliferation and increased L-lactate production of dermal fibroblasts in the GK-rat, a spontaneous model of non-insulin dependent diabetes mellitus.

Intact fibroblast function is required for normal wound healing. Although healing is generally accepted to be disturbed in non-insulin dependent diabetes mellitus, the signals modulating this disturbance are not fully understood. Therefore, we studied dermal fibroblasts from the GK rat, a non-insulin dependent diabetes mellitus model, and the Wistar rat (control) regarding growth characteristics, and L-lactate production at 5.5 mM and 25.5 mM glucose in the absence or presence of protein kinase C-inhibition, or alpha-tocopherol acetate. In addition, growth and L-lactate responses to hyaluronic acid were assessed under normal glucose conditions. At 5.5 mM glucose, the fibroblasts from the GK rat showed a lower proliferation rate during the first 24 hours, measured as DNA content, as compared to Wistar rats, i.e. at 8 hours GK was 57% of control, p < 0.01, at 24 hours GK was 60% of control, p < 0.01. The GK rat fibroblasts accumulated higher L-lactate levels in the media at 24-96 hours. Addition of glucose at a concentration of 25.5 mM decreased the total DNA content in GK rat fibroblast cultures to 74% (p < 0.05) and in control to 87% (p < 0.05), and increased L-lactate levels, measured at 48 hours. A protein kinase C-inhibitor, bisindolylmaleimide IX, increased DNA content and decreased L-lactate in both cell types during culture in high glucose, but only affected GK rat fibroblasts during normal glucose. Hyaluronic acid, increased DNA content in both types of fibroblasts, GK: 139% (p < 0.05), control: 127% (p < 0.05) and reduced L-lactate production. The above observations indicate that GK rat fibroblast proliferation is suppressed when the cells are cultured in high glucose containing media. In addition, protein kinase C and hyaluronic acid might play a role as modulators of fibroblast proliferation during the diabetic state.

Inhibited proliferation of fibroblasts derived from chronic diabetic wounds and normal dermal fibroblasts treated with high glucose is associated with increased formation of l-lactate.

Diabetes is accompanied by delayed wound healing and insufficient granulation tissue formation, possibly because of a defect in fibroblast function. We have previously shown that fibroblasts derived from chronic diabetic foot ulcers have lower proliferation compared with those from uninjured skin. The aim of this study was to investigate possible mechanisms explaining the impaired fibroblast proliferation observed in fibroblasts from non-insulin-dependent diabetes mellitus chronic wounds and normal fibroblasts cultured in high glucose. Fibroblasts from two groups of patients were studied: nondiabetic patients with chronic venous stasis ulcers and non-insulin-dependent diabetes mellitus patients with chronic diabetic wounds. Biopsies from both uninjured skin and wounds were taken from the same patients to serve as sources of fibroblasts. A fluorometric method was used to determine DNA content, and a spectrophotometric lactate oxidase method was used for lactate level analysis. We found a dose-dependent inhibition of normal fibroblast proliferation when adding conditioned media from non-insulin-dependent diabetes mellitus wound fibroblasts. The conditioned medium, from these cells showed elevated l-lactate levels, 6.3 +/- 0.7 mmol/L, compared with media derived from nondiabetic, 2.1 +/- 0.3 mmol/L (p < 0.01), and diabetic uninjured skin fibroblasts, 3.5 +/- 0.6 mmol/L, and from chronic nondiabetic wound fibroblasts 2.9 +/- 0.3 mmol/L. Addition of 6 mmol/L l-lactate to uninjured normal fibroblasts resulted in decreased DNA content (58 +/- 7%, p < 0.01). Previously we have shown that high glucose concentrations inhibit fibroblast proliferation and induce growth factor resistance. When increasing the amount of d-glucose in the media, l-lactate levels increased in all cell types. When the uninjured normal cells were treated with beta-hydroxybutyrate, the total DNA content decreased by 42 +/- 5% (p < 0.05), with no significant increase in the l-lactate levels. These observations indicate that l-lactate production may be of importance for fibroblast proliferation in vitro and may play a role in fibroblast proliferation in vivo.

Fibroblasts derived from human chronic diabetic wounds have a decreased proliferation rate, which is recovered by the addition of heparin.

We have studied the growth kinetics of fibroblasts derived from uninjured skin and chronic wounds in non-diabetic and diabetic (IDDM) patients. DNA measurements during the first 24 h after cell starvation showed that fibroblasts derived from chronic wounds, both non-diabetic and diabetic, display a decreased adhesion and proliferation. When determining the rate of proliferation after another 48, 72 and 96 h, a significant decrease in the proliferation rate was found in the chronic wound fibroblasts compared to those from uninjured skin. Furthermore, we have investigated the effects of heparin, hyaluronic acid and other heparin-like substances on the proliferation of non-diabetic and diabetic fibroblasts. We found that these substances stimulated the proliferation of human fibroblasts derived from both normal skin and chronic wounds measured as DNA content. Stimulation with heparin normalized the proliferation of the diabetic chronic wound fibroblasts. This effect was independent of the presence of serum. The effect of heparin was dose-dependent and most pronounced during the first 24 h of stimulation. These results suggest that heparin may be of importance in the treatment of chronic diabetic wounds.

High glucose-induced growth factor resistance in human fibroblasts can be reversed by antioxidants and protein kinase C-inhibitors.

We have studied the influence of high glucose on basal fibroblast proliferation, growth factor induced cellular proliferation and the effects of antioxidants, protein kinase C-inhibitors and troglitazone. Fibroblast cultures were obtained from five patients undergoing mammary reduction plastic surgery. A fluorometric method was used for determining total DNA in the cell samples, DNA content being proportional to cell number. D-Glucose at 15.5 mM and above was shown to inhibit fibroblast proliferation, and the cells were resistant to growth factors such as IGF-I and EGF at this glucose concentration. H7, bisindolylmaleimide IX, troglitazone, alpha-tocopherol acetate, Q10, ascorbic acid, beta-carotene, DMTU and selenite were all found to reverse the high glucose-induced growth factor resistance observed in human fibroblasts. We believe that these findings may be of value in the understanding and future treatment of wound healing in diabetic foot ulcers.

Long-term treatment of Swiss 3T3 fibroblasts with dexamethasone attenuates MAP kinase activation induced by insulin-like growth factor-I (IGF-I).

Bone formation is reduced in hyperglucocorticoid states, e.g. Cushing's syndrome or long-term treatment with synthetic glucocorticoids during rheumatic diseases. possibly related to decreased sensitivity of the target to insulin-like growth factor-I (IGF-I). In this study, we have sought to identify postreceptor-mechanisms for glucocorticoid-induced resistance to insulin-like peptides in a model system. Treatment of Swiss 3T3 fibroblasts with 100 nM dexamethasone for 48h reduced IGF-I-induced activation of mitogen-activated protein kinase (MAP kinase). The level of insulin receptor substrate-1 (IRS-1) was reduced in dexamethasone-treated cells, as measured by Western blot; however, the pattern of tyrosine-phosphorylated protein subsequent to stimulation with IGF-I (1 min) was not altered. No inhibitory effect of dexamethasone was observed on the level of phosphotyrosine in IRS-1 in extracts from IGF-I-treated cells. The amount of IGF-I-induced association of insulin receptor substrate-1 and phosphatidylinositol 3-kinase was increased in steroid treated cells. Addition of IGF-I increased the synthesis of lipid, glycogen and protein, and the reduction of a tetrazolium dye, MTS, in untreated cells. The response to IGF-I in terms of glycogen synthesis was blunted, whereas the effect of IGF-I was unaffected for the other three parameters in cells pretreated with dexamethasone. These findings indicate that the activation of MAP kinase may be dissociated from IGF-I-induced anabolic pathways and tyrosine phosphorylation of IRS-1. The results agree with the previously proposed role for the activation of MAP kinase in the regulation of glycogen synthesis. Furthermore, they suggest that dexamethasone-induced reduction of IRS-1 expression may be important for the impaired activation of MAP kinase by insulin-like peptides in steroid-treated cells.