Insulin-degrading enzyme (IDE) is a zinc metalloprotease that hydrolyzes amyloid-␤ (A␤) and insulin, which are peptides associated with Alzheimer disease (AD) and diabetes, respectively. Our previous structural analysis of substratebound human 113-kDa IDE reveals that the N-and C-terminal domains of IDE, IDE-N and IDE-C, make substantial contact to form an enclosed catalytic chamber to entrap its substrates. Furthermore, IDE undergoes a switch between the closed and open conformations for catalysis. Here we report a substrate-free IDE structure in its closed conformation, revealing the molecular details of the active conformation of the catalytic site of IDE and new insights as to how the closed conformation of IDE may be kept in its resting, inactive conformation. We also show that A␤ is degraded more efficiently by IDE carrying destabilizing mutations at the interface of IDE-N and IDE-C (D426C and K899C), resulting in an increase in V max with only minimal changes to K m . Because ATP is known to activate the ability of IDE to degrade short peptides, we investigated the interaction between ATP and activating mutations. We found that these mutations rendered IDE less sensitive to ATP activation, suggesting that ATP might facilitate the transition from the closed state to the open conformation. Consistent with this notion, we found that ATP induced an increase in hydrodynamic radius, a shift in electrophoretic mobility, and changes in secondary structure. Together, our results highlight the importance of the closed conformation for regulating the activity of IDE and provide new molecular details that will facilitate the development of activators and inhibitors of IDE.

Dietary AGEs detrimentally modulate gut microbial ecology and may partially increase colon permeability, which can adversely impact host health.

BackgroundInsulin-degrading enzyme (IDE) is widely recognized as the principal protease responsible for the clearance and inactivation of insulin, but its role in glycemic control in vivo is poorly understood. We present here the first longitudinal characterization, to our knowledge, of glucose regulation in mice with pancellular deletion of the IDE gene (IDE-KO mice).MethodologyIDE-KO mice and wild-type (WT) littermates were characterized at 2, 4, and 6 months of age in terms of body weight, basal glucose and insulin levels, and insulin and glucose tolerance. Consistent with a functional role for IDE in insulin clearance, fasting serum insulin levels in IDE-KO mice were found to be ∼3-fold higher than those in wild-type (WT) controls at all ages examined. In agreement with previous observations, 6-mo-old IDE-KO mice exhibited a severe diabetic phenotype characterized by increased body weight and pronounced glucose and insulin intolerance. In marked contrast, 2-mo-old IDE-KO mice exhibited multiple signs of improved glycemic control, including lower fasting glucose levels, lower body mass, and modestly enhanced insulin and glucose tolerance relative to WT controls. Biochemically, the emergence of the diabetic phenotype in IDE-KO mice correlated with age-dependent reductions in insulin receptor (IR) levels in muscle, adipose, and liver tissue. Primary adipocytes harvested from 6-mo-old IDE-KO mice also showed functional impairments in insulin-stimulated glucose uptake.ConclusionsOur results indicate that the diabetic phenotype in IDE-KO mice is not a primary consequence of IDE deficiency, but is instead an emergent compensatory response to chronic hyperinsulinemia resulting from complete deletion of IDE in all tissues throughout life. Significantly, our findings provide new evidence to support the idea that partial and/or transient inhibition of IDE may constitute a valid approach to the treatment of diabetes.

Thermally processed diets are widely consumed, although advanced-glycation end products (AGEs) are unavoidably formed. AGEs, clusters of protein-cross-linking products, become less digestible because they impair intestinal peptidase proteolysis. We characterized the impacts of dietary AGEs on gut microbiota through a microbiome-to-metabolome association study. C57BL/6 mice were fed a heat-treated diet (high-AGE diet, H-AGE) or a standard AIN-93G diet (low-AGE diet, L-AGE) for 8 months. Fecal-microbiota composition was examined by 16S rDNA sequencing, and fecal-metabolome profile was evaluated by gas chromatography-tandem time-of-flight mass spectrometry (GC-TOF-MS). Reduced α-diversity and altered microbiota composition with elevated Helicobacter levels were found in the H-AGE group, and among the 57 perturbed metabolites, protein-fermentation products (i.e., p-cresol and putrescine) were increased. Major dysfunctional metabolic pathways were associated with carbohydrate and amino acid metabolism in two groups. Moreover, high correlations were found between fluctuant gut microbiota and metabolites. These findings might reveal the underlying mechanisms of the detrimental impacts of dietary AGEs on host health.

Insulin-degrading enzyme (IDE) is a ubiquitously expressed zinc-metalloprotease that degrades several pathophysiologically significant extracellular substrates, including insulin and the amyloid β-protein (Aβ), and accumulating evidence suggests that IDE dysfunction may be operative in both type 2 diabetes mellitus and Alzheimer disease (AD). Although IDE is well known to be secreted by a variety of cell types, the underlying trafficking pathway(s) remain poorly understood. To address this topic, we investigated the effects of known inhibitors or stimulators of protein secretion on the secretion of IDE from murine hepatocytes and HeLa cells. IDE secretion was found to be unaffected by the classical secretion inhibitors brefeldin A (BFA), monensin, or nocodazole, treatments that readily inhibited the secretion of α1-antitrypsin (AAT) overexpressed in the same cells. Using a novel cell-based Aβ-degradation assay, we show further that IDE secretion was similarly unaffected by multiple stimulators of protein secretion, including glyburide and 3'-O-(4-benzoyl)benzoyl-ATP (Bz-ATP). The calcium ionophore, A23187, increased extracellular IDE activity, but only under conditions that also elicited cytotoxicity. Our results provide the first biochemical evidence that IDE export is not dependent upon the classical secretion pathway, thereby identifying IDE as a novel member of the select class of unconventionally secreted proteins. Further elucidation of the mechanisms underlying IDE secretion, which would be facilitated by the assays described herein, promises to uncover processes that might be defective in disease or manipulated for therapeutic benefit. ResultsAccumulating evidence from cell and animal modeling studies and human molecular genetics implicates impaired function of IDE in the pathogenesis of type 2 diabetes mellitus and Alzheimer disease (AD) [1][2][3]. IDE is the prototypical member of an evolutionarily distinct superfamily of zinc-metalloproteases possessing several features that distinguish it from conventional metalloproteases, including an "inverted" zinc-binding motif (HxxEH) [4] and an unusual tertiary structure [5][6][7]. Another distinguishing feature of IDE is its subcellular localization: the vast majority of IDE is present in the cytosol, with smaller amounts present in mitochondria, peroxisomes, and endosomes [8]. A small fraction of IDE-estimated to be 3% to 10% of the total-is also trafficked to the extracellular space, and it is this pool which interacts with known substrates of IDE, such as insulin and Aβ [9]. Despite a large number of studies demonstrat-