Page 77 - Biennial Report 2018-20 Jun 2021

seeded in six well plates and were allowed to grow upto 70-80% confluency and treated with
glucose (25 mM for 6 and 12h). Real-time PCR for IGF-1 was performed and 18S rRNA used as
control. Glucose caused a significant increase in mRNA levels of IGF-1. Although an increased
trend is evident at 6h, the effect is more pronounced and significant at 12h. The role of IGF-1
signaling was further assessed in HaCaT cells initiated via the glucose trigger in WS1 at 12h.
Fibroblasts (WS1) were seeded onto the insert and keratinocytes (HaCaT) on the bottom
chamber in co-culture plates. The cells were serum starved for 12 hrs prior and treated with high
glucose (25mM). On termination of incubation at 12h, p-Akt, an important intermediate of IGF-
1 signaling was assessed in HaCaT cells. There was a significant alteration in the levels of p-Akt
in keratinocytes upon glucose treatment (25mM) in WS1 cells. However, there was no change in
the total levels of Akt, suggesting that glucose treatment in WS1 cells caused release of some
factor(s) that altered Akt phosphorylation in keratinocytes. Also, when WS1 fibroblast cells were
treated with high glucose (25mM) for 12h, this caused the mitochondria to become round with
irregular cristae formation similar to what was seen in keratinocytes. However, in normal WS1
cells, the mitochondria appeared elongated with regular cristae. Ultrastructural studies for all
diabetic human skin samples were completed and histological findings suggest that diabetic skin
shows higher number of blood vessels in dermis together with the presence of Melanophages in
the dermis.

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