A pathological feature of Type 2 diabetes is deposits in the pancreatic islets primarily composed of amylin (islet amyloid polypeptide). Although much attention has been paid to the expression and secretion of amylin, little is known about the enzymes involved in amylin turnover. Recent reports suggest that insulin-degrading enzyme (IDE) may have specificity for amyloidogenic proteins, and therefore we sought to determine whether amylin is an IDE substrate. Amylin-degrading activity co-purified with IDE from rat muscle through several chromatographic steps. Metalloproteinase inhibitors inactivated amylin-degrading activity with a pattern consistent with the enzymatic properties of IDE, whereas inhibitors of acid and serine proteases, calpains, and the proteasome were ineffective. Amylin degradation was inhibited by insulin in a dose-dependent manner, whereas insulin degradation was inhibited by amylin. Other substrates of IDE such as atrial natriuretic peptide and glucagon also competitively inhibited amylin degradation. Radiolabeled amylin and insulin were both covalently cross-linked to a protein of 110 kDa, and the binding was competitively inhibited by either unlabeled insulin or amylin. Finally, a monoclonal anti-IDE antibody immunoprecipitated both insulin- and amylin-degrading activities. The data strongly suggest that IDE is an amylin-degrading enzyme and plays an important role in the clearance of amylin and the prevention of islet amyloid formation.

Relaxin activation of its receptor RXFP1 triggers multiple signaling pathways. Previously, we have shown that relaxin activates PPARγ transcriptional activity in a ligand-independent manner, but the mechanism for this effect was unknown. In this study, we examined the signaling pathways of downstream of RXFP1 leading to PPARγ activation. Using cells stably expressing RXFP1, we found that relaxin regulation of PPARγ activity requires accumulation of cAMP and subsequent activation of cAMP-dependent protein kinase (PKA). The activated PKA subsequently phosphorylated cAMP response element-binding protein (CREB) at Ser-133 to activate it directly, as well as indirectly through mitogen activated protein kinase p38 MAPK. Activated CREB was required for relaxin stimulation of PPARγ activity, while there was no evidence for a role of the nitric oxide or ERK MAPK pathways. Relaxin increased the mRNA and protein levels of the coactivator protein PGC1α, and this effect was dependent on PKA, and was completely abrogated by a dominant-negative form of CREB. This mechanism was confirmed in a hepatic stellate cell line stably that endogenously expresses RXFP1. Reduction of PGC1α levels using siRNA diminished the regulation of PPARγ by relaxin. These results suggest that relaxin activates the cAMP/PKA and p38 MAPK pathways to phosphorylate CREB, resulting in increased PGC1α levels. This provides a mechanism for the ligand-independent activation of PPARγ in response to relaxin.Relaxin activates peroxisome proliferator-activated receptor γ (PPARγ) by an unknown PPARγ ligand-independent mechanism. Results Expression of PPARγ coactivator 1α (PGC1α) was increased by relaxin through distinct signaling pathways. Conclusion The mechanism for relaxin regulation of PPARγ is through increased PGC1α levels. Significance Discovery of new targets of relaxin signaling may provide further understanding of its pleiotropic effects.

A consequence of insulin-dependent diabetes mellitus is the loss of lean muscle mass as a result of accelerated proteolysis by the proteasome. Insulin inhibition of proteasomal activity requires interaction with insulin-degrading enzyme (IDE), but it is unclear if proteasome inhibition is dependent merely on insulin-NIDE binding or if degradation of insulin by IDE is required. To test the hypothesis that degradation by IDE is required for proteasome inhibition, a panel of insulin analogues with variable susceptibility to degradation by IDE binding was used to assess effects on the proteasome. The analogues used were [Lys(B28), Pro(B29)]-insulin (lispro), [Asp(B10)]-insulin (Asp(B10)) and [Glu(B4), Gln(B16), Phe(B17)]-insulin (EQF). Lispro was as effective as insulin at inhibition of degradation of iodine-125 ((125)I)-labeled insulin, but Asp(B10) and EQF were somewhat more effective. All agents inhibited cross-linking of (125)I-insulin to IDE, suggesting that all were capable of IDE binding. In contrast, although insulin and lispro were readily degraded by IDE, Asp(B10) was degraded more slowly, and EQF degradation was undetectable. Both insulin and lispro inhibited the proteasome, but Asp(B10) was less effective, and EQF had little effect. In summary, despite effective IDE binding, EQF was poorly degraded by IDE, and was ineffective at proteasome inhibition. These data suggest that insulin inhibition of proteasome activity is dependent on degradation by IDE. The mechanism of proteasome inhibition may be the generation of inhibitory fragments of insulin, or by displacement of IDE from the proteasome.