Previous work has shown that insulin improves wound healing, but the mechanisms of its action on healing have not been delineated. We used a variety of cell and molecular approaches to determine the effects of insulin on cutaneous wounds and have shown that insulin: (1) stimulates keratinocyte migration in a dose- and time-dependent manner; (2) acts in an insulin-receptor-dependent but EGF/EGF-R-independent manner; (3) stimulates keratinocyte migration through the PI3K-Akt-Rac1 pathway; (4) stimulates keratinocytes to produce integrin α3 and LN332, and cell migration in vitro and in vivo is dependent on these molecules. The ability of this hormone to stimulate a variety of cell functions important for wound healing in an insulin-receptor-dependent manner creates the ability to target processes that are dependent only on insulin. Conversely, the ability to stimulate both the insulin and IGF-1 receptors may broaden the applicability of insulin in different wound conditions, particularly when one receptor may be missing or dysfunctional (e.g. type II diabetes).
Understanding the processes by which insulin accelerates wound closure is important because it will provide insight into potential manipulation of the healing process using this hormone as well as the signaling molecules it activates. The results presented here show that stimulation of keratinocyte migration by insulin involves the PI3K-Akt pathway, and identifies Rac1, a small GTPase, as a molecule activated downstream of PI3K-Akt. Support for these conclusions includes the fact that these molecules are activated upon insulin stimulation, and inhibition of each molecule with specific inhibitors or dominant negative proteins prevents insulin-induced keratinocyte migration.
Rac1 is known to regulate actin assembly  and to stimulate formation of lamellipodia , thereby promoting cell movement in response to external signals from cytokines, growth factors and/or the ECM. Our results support insulin-induced Rac1 activation in keratinocytes, as shown by Rac1 translocation from the cytosol to the cell membrane, the formation of membrane ruffles upon insulin stimulation, and the increased levels of active Rac1 identified by the pull down assay. Moreover, we show that Rac1 activation is dependent on the PI3K-Akt signaling pathway in keratinocyte migration. Two recent papers showed a strong activation of the PI3K pathway in the wound margin keratinocytes [28, 29]. Furthermore, epidermal growth factor (EGF) or heregulin (HRG) have been found to stimulate keratinocyte migration through PI3K/Akt signaling . These results, coupled with our findings presented here, show that insulin can replace some well known growth factors in wound healing.
The effects of insulin on keratinocyte migration described above, led us to hypothesize that insulin-accelerated wound healing involves increased expression of the integrin α3β1 in keratinocytes as well as an increase in the levels of LN332. The latter protein is a matrix molecule secreted by migrating keratinocytes at the leading edge , where it mediates keratinocyte polarity and persistent migration [32, 33]. After wounding, quiescent epidermal keratinocytes are activated and express the integrins α6β4 and α3β1, which mediate their migration on LN332 and facilitate the development of the BM . Our results show that insulin stimulates keratinocyte integrin α3 expression and LN332 deposition, and that inhibition of these proteins in vitro or in vivo inhibits insulin-induced keratinocyte migration and wound healing, strongly suggesting an important role for these molecules in insulin stimulation of healing. This is a particularly important finding, as the relationship between LN332 and migration has been controversial. Indeed, previous studies have implicated LN332 in promoting and inhibiting cell migration [34–36]; our results support a role for LN332 in promoting keratinocyte migration.
In addition to its effects on keratinocyte migration during wound healing, insulin also promotes attachment of the epidermis to the dermis, the appearance of a well-organized epidermis, increased numbers of skin appendages, and more dermal papilla and epidermal reticular ridges. We also found that inhibiting integrin α3 and LN332 resulted in lack of full epidermal differentiation (as shown by disorganization of the epidermis) and decreased formation of dermal papilla and epidermal reticular ridges.
Our results suggest that the direct application of insulin to chronic wounds may improve wound healing by compensating for a deficiency of insulin and/or IGF-1 in the injured area. Previous studies have shown that the IGF-1 level is decreased in both streptozotocin (STZ)-induced diabetic and normal rat incision wounds , and we have also detected a decrease in insulin levels in rat scald wounds (unpublished data). This insulin and IGF-1 deficiency in the wound, along with our data showing that insulin can function through stimulation of both insulin and IGF-1 receptors, suggests that direct application of insulin to the wound area may improve healing through activation of both receptors. Indeed, it has been shown that topical application of insulin accelerates healing of infected cutaneous ulcerations in diabetic mice , showing that insulin is promising for treatment of these types of wounds. However, it has also been shown that leptin-deficient obese/obese (ob/ob) diabetic mice have dysfunctional signaling during wound healing , suggesting that in these mice mediating the effect of insulin on wounds might be different from those in normal wounds or wounds in diabetic people.
When compared with other growth factors used to promote wound repair, insulin treatment is likely to be much less expensive, more readily available, and has already been approved by the FDA for human use. Moreover, a significant numbers of reports have described that treatment with growth factors, including most of the growth factors that have been used clinically, or increased expression of growth factor receptors, lead to carcinogenesis [39–41]. In contrast, insulin is safe, as shown by its use for nearly a hundred years, hence it is likely safer than growth factor alternatives. When choosing a concentration of insulin for possible wound therapy, it is important to remember that, although the highest concentrations of insulin resulted in the greatest keratinocyte migration, such doses may alter blood glucose levels in vivo.