Monogenic forms of permanent neonatal diabetes and type 2 -cell decompensation have highlighted the importance and vulnerability of proinsulin processing in the endoplasmic reticulum (ER). Once thought to be housekeeping functions, the processes of proinsulin folding, ER associated protein degradation (ERAD)- mediated quality and quantity controand trafficking of proinsulin from the ER to the Golgi are all highly regulated as a function of the metabolic state and demand for insulin. We have identified the PERK eIF2a kinase, whose loss of function results in permanent neonatal diabetes in the Wolcott Rallison syndrome, as a key regulator of proinsulin processing in the ER. We postulated a novel and controversial model that PERK regulates proinsulin processing by controlling the expression of key ER chaperone and folding proteins including GRP78 and ERp72, which, in turn, control whether proinsulin is degraded by the ERAD pathway or trafficked forward to the Golgi for proteolytic processing and packaging into secretory granules. In this proposal we will further test this model by manipulating the PERK-eIF2a pathway and the expression of key ER chaperones and assessing the fate and processing state of proinsulin. We will also determine how PERK- eIF2a modulates proinsulin processing in response to fluctuating glucose levels in order to provide the appropriate level of insulin production. Based on preliminary results that Guanabenz, an eIF2a dephosphorylation inhibitor, can reverse the severe proinsulin aggregation seen in PERK deficient -cells, we proposed to test its ability to prevent or reverse the severe permanent neonatal diabetes associated with the Wolcott Rallison Syndrome and dominant-negative acting insulin mutants (MIDY). This strategy may also be applicable to treatment of -cell dysfunctions associated with type 2 diabetes decompensation.
The final Aim i s designed to identify the molecular mechanisms underlying PERK-dependent regulation of ER chaperones and is essential for understanding the regulatory context in which PERK plays a central role in regulating -cell functions.
Maintaining a narrow range of circulating insulin is critical to ensuring normal blood glucose levels and preventing the onset of diabetes and it's plethora of negative downstream effects on human health. Although the basic mechanisms underlying the tight control of insulin synthesis, capacity, and secretion have been investigated intensely, the mechanisms by which these processes are integrated to ensure that the right amount of insulin is maintained in circulation are poorly understood. Our previous studies implicate the PERK eIF2a kinase as a critical coordinator of insulin folding, quality and quantity control, traffickin and secretion, and the Aims described in this proposal are designed to uncover the mechanisms by which this important regulation is achieved, and apply these discoveries to the treatment of permanent neonatal diabetes.
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