**3. Diabetes and IRBP levels**

Considering visual cycle components, decreased IRBP expression is one of the most characteristic changes in diabetes. Many studies have evaluated the changes in protein levels and IRBP expression and also attempted to explain the reasons for its depletion.

One study revealed decreased expression of IRBP determined by both qPCR and protein quantification on post-mortem samples of diabetic patients [28]. Another study showed that this decreased expression directly correlated with the evolution of the DR, and also tested the effect of glucose and inflammatory cytokines on IRBP expression *in vitro*. They found that high glucose, TNF-α and IL-1β were able to reduce IRBP's expression [29]. A recent study found decreased IRBP levels in diabetic rats and this finding was accompanied by decreased levels of 11-cRAL and rhodopsin synthesis [30].

The precise mechanisms responsible for the decreased IRBP levels remain unclear. It is known that high glucose and some circulating fatty acids can induce the secretion of inflammatory cytokines by Müller cells [31, 32]. Despite evidence that high levels of glucose and inflammatory cytokines are able to decrease the expression of IRBP [24, 29], other mechanisms may be involved. With the early onset of diabetes, photoreceptors undergo oxidative stress resulting in increased nitrosative-oxidative stress [33, 34]. This biochemical stress can induce damage to proteins promoting their degradation [35]. The unfolded protein response (UPR) has been detected to be active in photoreceptor cells in animal studies [36]; however no studies have linked this process to decreased IRBP levels.

Disruption of the external limiting membrane (ELM) and the outer retinal barrier (ORB) may play a role in leaking of IRBP into the outer nuclear layer or Bruch's membrane. Studies of animals in diabetic conditions have shown decreased occluding abilities in the Müller cell tight junctions compromising the external limiting membrane [37]. Also retinal pigment epithelium (RPE) dysfunction in early stage diabetes has been described in animal models [38]. It is still unclear the impact of these mechanisms over the IRBP levels.
