Evolutionary Origin of Insulin-Degrading Enzyme and Its Subcellular Localization and Secretion Mechanism: A Study in Microglial Cells

The insulin-degrading enzyme (IDE) is a zinc-dependent metalloendopeptidase that belongs to the M16A metalloprotease family. IDE is markedly expressed in the brain, where it is particularly relevant due to its in vitro amyloid beta (Aβ)-degrading activity. The subcellular localization of IDE, a paramount aspect to understand how this enzyme can perform its proteolytic functions in vivo, remains highly controversial. In this work, we addressed IDE subcellular localization from an evolutionary perspective. Phylogenetic analyses based on protein sequence and gene and protein structure were performed. An in silico analysis of IDE signal peptide suggests an evolutionary shift in IDE exportation at the prokaryote/eukaryote divide. Subcellular localization experiments in microglia revealed that IDE is mostly cytosolic. Furthermore, IDE associates to membranes by their cytoplasmatic side and further partitions between raft and non-raft domains. When stimulated, microglia change into a secretory active state, produces numerous multivesicular bodies and IDE associates with their membranes. The subsequent inward budding of such membranes internalizes IDE in intraluminal vesicles, which later allows IDE to be exported outside the cells in small extracellular vesicles. We further demonstrate that such an IDE exportation mechanism is regulated by stimuli relevant for microglia in physiological conditions and upon aging and neurodegeneration.

Ginsenoside F1 Protects the Brain against Amyloid Beta-Induced Toxicity by Regulating IDE and NEP

Ginsenoside F1, the metabolite of Rg1, is one of the most important constituents of Panax ginseng. Although the effects of ginsenosides on amyloid beta (Aβ) aggregation in the brain are known, the role of ginsenoside F1 remains unclear. Here, we investigated the protective effect of ginsenoside F1 against Aβ aggregation in vivo and in vitro. Treatment with 2.5 μM ginsenoside F1 reduced Aβ-induced cytotoxicity by decreasing Aβ aggregation in mouse neuroblastoma neuro-2a (N2a) and human neuroblastoma SH-SY5Y neuronal cell lines. Western blotting, real-time PCR, and siRNA analysis revealed an increased level of insulin-degrading enzyme (IDE) and neprilysin (NEP). Furthermore, liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis confirmed that ginsenoside F1 could pass the blood–brain barrier within 2 h after administration. Immunostaining results indicate that ginsenoside F1 reduces Aβ plaques in the hippocampus of APPswe/PSEN1dE9 (APP/PS1) double-transgenic Alzheimer’s disease (AD) mice. Consistently, increased levels of IDE and NEP protein and mRNA were observed after the 8-week administration of 10 mg/kg/d ginsenoside F1. These data indicate that ginsenoside F1 is a promising therapeutic candidate for AD.

Genetic Inactivation of Free Fatty Acid Receptor 3 Impedes Behavioral Deficits and Pathological Hallmarks in the APP Swe Alzheimer’s Disease Mouse Model

The free fatty acid FFA3 receptor (FFA3R) belongs to the superfamily of G-protein-coupled receptors (GPCRs). In the intestine and adipose tissue, it is involved in the regulation of energy metabolism but its function in the brain is unknown. We aimed, first, to investigate the expression of the receptor in the hippocampus of Alzheimer disease (AD) patients at different stages of the disease and, second, to assess whether genetic inactivation of the Ffar3 gene could affect the phenotypic features of the APPswe mouse model. The expression of transcripts for FFA receptors in post mortem human hippocampal samples and in the hippocampus of wild-type and transgenic mice was analyzed by RT-qPCR. We generated a double transgenic mouse, FFA3R-/-/APPswe, to perform cognition studies and to assess, by immunoblotting, Aβ and tau pathologies and the differential expression of synaptic plasticity-related proteins.For the first time, the occurrence of the FFA3R in the human hippocampus and its overexpression, even in the first stages of AD, was demonstrated. Remarkably, FFA3R-/-/APPswe mice do not have the characteristic memory impairment of 12-month-old APPswe mice. Also, this newly generated transgenic line does not develop the most important Alzheimer’s disease (AD)-related features, such as amyloid beta (Aβ) brain accumulations and tau hyperphosphorylation. These findings are accompanied by increased levels of the insulin-degrading enzyme (IDE) and lower activity of the tau kinases GSK3β and Cdk5. We conclude that the brain FFA3R is involved in cognitive processes and its inactivation prevents AD-like cognitive decline and pathological hallmarks.

ANTIOXIDATIVE PROPERTIES OF GEROPROTECTIVE PEPTIDES

Приведенные в работе результаты исследований об антиоксидантном действии ряда пептидных препаратов, которое наблюдается на различных уровнях, начиная от клеточного до организма в целом, свидетельствуют о важной роли низкомолекулярных пептидов в механизмах регуляции гомеостаза при старении. Антиоксидантные свойства регуляторных пептидов проявляются в экспериментах как на интактных половозрелых животных, так и особенно наглядно при старении или действии экстремальных факторов внешней среды (гипоксия, гипокинезия). Ряд исследуемых пептидов (AEDG, KE) оказывает мембраностабилизирующее действие, препятствуя осмотическому и кислотному гемолизу эритроцитов и снижая уровень содержания внеэритроцитарного гемоглобина и суммарной пероксидазной активности в плазме крови. Показано, что исследуемые пептиды способны оказывать нейропротекторный эффект путем стабилизации активности ферментов (неприлизин, инсулиндеградирующий фермент), играющих важную роль в катаболизме β-амилоида, и препятствовать его накоплению в мозге. Рассматривается участие регуляторных пептидов, обладающих антиоксидантными свойствами, на экспрессию генов, обеспечивающих стабилизацию митохондриальных мембран, функционирование электрон-транспортной цепи и активность антиоксидантных ферментов. The results of studies on the antioxidant effect of a number of peptide drugs, which is manifested at various levels, ranging from cells to the whole body, in adulthood and during aging, under the influence of extreme environmental factors, indicate the important role of low-molecular peptides in the mechanisms of regulating homeostasis during aging. The antioxidant properties of regulatory peptides are shown in experiments both on intact sexually mature animals, and especially clearly during aging or the action of extreme environmental factors (hypoxia, hypokinesia). A number of the studied substances (AEDG, KE) have a membrane-stabilizing effect, preventing osmotic and acid hemolysis of red blood cells and reducing the level of extra-erythrocyte hemoglobin and total peroxidase activity in blood plasma. It is shown that the studied peptides are able to have a neuroprotective effect by stabilizing the activity of enzymes (neprilysin, an insulin degrading enzyme) that play an important role in the catabolism of beta-amyloid, and prevent its accumulation in concentrations toxic to cells. The involvement of regulatory peptides with antioxidant properties on the expression of genes that ensure the stabilization of mitochondrial membranes, the functioning of the electron transport chain and the activity of antioxidant enzymes is considered.

Medium-Chain Length Fatty Acids Enhance Aβ Degradation by Affecting Insulin-Degrading Enzyme

The accumulation of amyloid β-protein (Aβ) is one of the major pathological hallmarks of Alzheimer’s disease. Insulin-degrading enzyme (IDE), a zinc-metalloprotease, is a key enzyme involved in Aβ degradation, which, in addition to Aβ production, is critical for Aβ homeostasis. Here, we demonstrate that saturated medium-chain fatty acids (MCFAs) increase total Aβ degradation whereas longer saturated fatty acids result in an inhibition of its degradation, an effect which could not be detected in IDE knock-down cells. Further analysis of the underlying molecular mechanism revealed that MCFAs result in an increased exosomal IDE secretion, leading to an elevated extracellular and a decreased intracellular IDE level whereas gene expression of IDE was unaffected in dependence of the chain length. Additionally, MCFAs directly elevated the enzyme activity of recombinant IDE, while longer-chain length fatty acids resulted in an inhibited IDE activity. The effect of MCFAs on IDE activity could be confirmed in mice fed with a MCFA-enriched diet, revealing an increased IDE activity in serum. Our data underline that not only polyunsaturated fatty acids such as docosahexaenoic acid (DHA), but also short-chain fatty acids, highly enriched, for example in coconut oil, might be beneficial in preventing or treating Alzheimer’s disease.

IL-6 induced enhanced clearance of proANP and ANP by insulin-degrading enzyme in T1DM mouse

Cardiovascular disease (CVD) is the prevalent cause of morbidity and mortality in type I diabetes mellitus (T1DM) worldwide. However, the pathophysiological mechanisms underlying the relationship between CVD, CVD risk factors, and T1DM have not yet been sufficiently explored. Here we reported that insulin-degrading enzyme (IDE) effectively degrades the precursor of atrial natriuretic peptide (proANP) intracellular in HEK293T cells. Pro-inflammatory cytokine IL-6 elicited a significant dose-dependent increase in IDE protein expression. Inhibition of ERK/MAPK signaling pathway with selumetinib abolished IL-6-stimulated increase in IDE protein level and deceased in ANP secretion in H9C2 cells. Importantly, the T1DM mouse model displayed lower proANP in the heart and ANP in serum, due to increased IDE expression and activity. Our outcomes suggest a novel role of IL-6 on ANP metabolism via IDE and provide the possibilities for new potential therapeutic strategies for diabetes-related cardiovascular complications.

Insulin-Degrading Enzyme Regulates the Proliferation and Apoptosis of Porcine Skeletal Muscle Stem Cells via Myostatin/MYOD Pathway

Identifying the genes relevant for muscle development is pivotal to improve meat production and quality in pigs. Insulin-degrading enzyme (IDE), a thiol zinc-metalloendopeptidase, has been known to regulate the myogenic process of mouse and rat myoblast cell lines, while its myogenic role in pigs remained elusive. Therefore, the current study aimed to identify the effects of IDE on the proliferation and apoptosis of porcine skeletal muscle stem cells (PSMSCs) and underlying molecular mechanism. We found that IDE was widely expressed in porcine tissues, including kidney, lung, spleen, liver, heart, and skeletal muscle. Then, to explore the effects of IDE on the proliferation and apoptosis of PSMSCs, we subjected the cells to siRNA-mediated knockdown of IDE expression, which resulted in promoted cell proliferation and reduced apoptosis. As one of key transcription factors in myogenesis, MYOD, its expression was also decreased with IDE knockdown. To further elucidate the underlying molecular mechanism, RNA sequencing was performed. Among transcripts perturbed by the IDE knockdown after, a downregulated gene myostatin (MSTN) which is known as a negative regulator for muscle growth attracted our interest. Indeed, MSTN knockdown led to similar results as those of the IDE knockdown, with upregulation of cell cycle-related genes, downregulation of MYOD as well as apoptosis-related genes, and enhanced cell proliferation. Taken together, our findings suggest that IDE regulates the proliferation and apoptosis of PSMSCs via MSTN/MYOD pathway. Thus, we recruit IDE to the gene family of regulators for porcine skeletal muscle development and propose IDE as an example of gene to prioritize in order to improve pork production.

Effects of Fasting and Feeding on Transcriptional and Posttranscriptional Regulation of Insulin-Degrading Enzyme in Mice

Insulin-degrading enzyme (IDE) is a highly conserved and ubiquitously expressed Zn2+-metallopeptidase that regulates hepatic insulin sensitivity, albeit its regulation in response to the fasting-to-postprandial transition is poorly understood. In this work, we studied the regulation of IDE mRNA and protein levels as well as its proteolytic activity in the liver, skeletal muscle, and kidneys under fasting (18 h) and refeeding (30 min and 3 h) conditions, in mice fed a standard (SD) or high-fat (HFD) diets. In the liver of mice fed an HFD, fasting reduced IDE protein levels (~30%); whereas refeeding increased its activity (~45%) in both mice fed an SD and HFD. Likewise, IDE protein levels were reduced in the skeletal muscle (~30%) of mice fed an HFD during the fasting state. Circulating lactate concentrations directly correlated with hepatic IDE activity and protein levels. Of note, L-lactate in liver lysates augmented IDE activity in a dose-dependent manner. Additionally, IDE protein levels in liver and muscle tissues, but not its activity, inversely correlated (R2 = 0.3734 and 0.2951, respectively; p < 0.01) with a surrogate marker of insulin resistance (HOMA index). Finally, a multivariate analysis suggests that circulating insulin, glucose, non-esterified fatty acids, and lactate levels might be important in regulating IDE in liver and muscle tissues. Our results highlight that the nutritional regulation of IDE in liver and skeletal muscle is more complex than previously expected in mice, and that fasting/refeeding does not strongly influence the regulation of renal IDE.

Insulin-Degrading Enzyme: Paradoxes and Possibilities

More than seven decades have passed since the discovery of a proteolytic activity within crude tissue extracts that would become known as insulin-degrading enzyme (IDE). Certainly much has been learned about this atypical zinc-metallopeptidase; at the same time, however, many quite fundamental gaps in our understanding remain. Herein, I outline what I consider to be among the most critical unresolved questions within the field, many presenting as intriguing paradoxes. For instance, where does IDE, a predominantly cytosolic protein with no signal peptide or clearly identified secretion mechanism, interact with insulin and other extracellular substrates? Where precisely is IDE localized within the cell, and what are its functional roles in these compartments? How does IDE, a bowl-shaped protein that completely encapsulates its substrates, manage to avoid getting “clogged” and thus rendered inactive virtually immediately? Although these paradoxes are by definition unresolved, I offer herein my personal insights and informed speculations based on two decades working on the biology and pharmacology of IDE and suggest specific experimental strategies for addressing these conundrums. I also offer what I believe to be especially fruitful avenues for investigation made possible by the development of new technologies and IDE-specific reagents. It is my hope that these thoughts will contribute to continued progress elucidating the physiology and pathophysiology of this important peptidase.