Insulin Degradation Research Articles

Familial diabetes predisposes PCOS patients to insulin resistance (IR), reproductive impairment and hepatic dysfunction: effects of d-chiro inositol (DCI) and alpha lipoic acid (ALA) administration on hepatic insulin extraction (HIE) index

Objective PCOS is a syndrome is characterized by 2 out of 3 of the criteria established during the Rotterdam Consensus Conference. Recently the issue of insulin resistance (IR) has caught attention. Subjects A group of overweight/obese PCOS patients (n = 30) have been evaluated before and after 3 months of daily integrative administration of d-chiro inositol (DCI) (500 mg) and alpha lipoic acid (ALA) (300 mg). Methods Hormonal and metabolic profiles, oral glucose tolerance test (OGTT) for glucose, insulin and C-peptide response were performed in baseline conditions and after DCI plus ALA treatment. Hepatic Insulin Extraction (HIE) index was computed along the OGTT to evaluate the liver ability in degrading insulin. Results The treatment decreased LH, Androstenedione (A), insulin plasma levels, BMI, HOMA index, AST and ALT. Considering patients for the presence (n = 17) or absence of familial diabetes (n = 13), the greatest improvements occurred in the former patients. Insulin response to OGTT was greatly reduced after the treatment interval in PCOS with familial diabetes. HIE computation disclosed that in presence of familial diabetes liver degradation of insulin is reduced thus leaving a higher amount of circulating insulin. DCI plus ALA administration decreased AST and ALT and restored hepatic insulin clearance since HIE profile was improved. Conclusion Our study demonstrates that in overweight/obese PCOS the predisposition to familial diabetes triggers IR not only through the endogenous impaired DCI and ALA synthesis but also through a reduced hepatic clearance of insulin. DCI plus ALA administration positively improved hormonal, metabolic profiles as well as liver function.

Insulin-Degrading Enzyme, an Under-Estimated Potential Target to Treat Cancer?

Insulin-degrading enzyme (IDE) is a multifunctional protease due to the variety of its substrates, its various cellular locations, its conservation between species and its many non-proteolytic functions. Numerous studies have successfully demonstrated its implication in two main therapeutic areas: metabolic and neuronal diseases. In recent years, several reports have underlined the overexpression of this enzyme in different cancers. Still, the exact role of IDE in the physiopathology of cancer remains to be elucidated. Known as the main enzyme responsible for the degradation of insulin, an essential growth factor for healthy cells and cancer cells, IDE has also been shown to behave like a chaperone and interact with the proteasome. The pharmacological modulation of IDE (siRNA, chemical compounds, etc.) has demonstrated interesting results in cancer models. All these results point towards IDE as a potential target in cancer. In this review, we will discuss evidence of links between IDE and cancer development or resistance, IDE’s functions, catalytic or non-catalytic, in the context of cell proliferation, cancer development and the impact of the pharmacomodulation of IDE via cancer therapeutics.

Antibody-Mediated Inhibition of Insulin-Degrading Enzyme Improves Insulin Activity in a Diabetic Mouse Model.

Diabetes is a metabolic disease that may lead to different life-threatening complications. While insulin constitutes a beneficial treatment, its use may be limited due to increased degradation and an increase in side effects such as weight gain and hypoglycemia. Small molecule inhibitors to insulin-degrading enzyme (IDE) have been previously suggested as a potential treatment for diabetes through their ability to reduce insulin degradation and thus increase insulin activity. Nevertheless, their tendency to bind to the zinc ion in the catalytic site of IDE may affect other important metalloproteases and limit their clinical use. Here, we describe the isolation of an IDE-specific antibody that specifically inhibits insulin degradation by IDE. Using phage display, we generated a human IDE-specific antibody that binds human and mouse IDE with high affinity and specificity and can differentiate between active IDE to a mutated IDE with reduced catalytic activity in the range of 30 nM. We further assessed the ability of that IDE-inhibiting antibody to improve insulin activity in vivo in an STZ-induced diabetes mouse model. Since human antibodies may stimulate the mouse immune response to generate anti-human antibodies, we reformatted our inhibitory antibody to a “reverse chimeric” antibody that maintained the ability to inhibit IDE in vitro, but consisted of mouse constant regions, for reduced immunogenicity. We discovered that one intraperitoneal (IP) administration of the IDE-specific antibody in STZ-induced diabetic mice improved insulin activity in an insulin tolerance test (ITT) assay and reduced blood glucose levels. Our results suggest that antibody-mediated inhibition of IDE may be beneficial on improving insulin activity in a diabetic environment.

Alpha-Glucosidase and Alpha-Amylase Inhibitory Activities, Molecular Docking, and Antioxidant Capacities of Plectranthus ecklonii Constituents.

Shortage in insulin secretion or degradation of produced insulin is the principal characteristic of the metabolic disorder of diabetes mellitus (DM). However, because the current medications for the treatment of DM have many detrimental side effects, it is necessary to develop more effective antidiabetic drugs with minimal side effects. Alpha-glucosidase and alpha-amylase inhibitors are directly implicated in the delay of carbohydrate digestion. Pharmacologically, these inhibitors could be targeted for the reduction in glucose absorption rate and, subsequently, decreasing the postprandial rise in plasma glucose and the risk for long-term diabetes complications. The main objectives of this research study were to isolate different phytochemical constituents present in the methanolic extract of Plectranthus ecklonii and evaluate their alpha-glucosidase and alpha-amylase inhibitory activities and antioxidant capacity. The phytochemical investigation of the methanolic extract of P. ecklonii yielded three known compounds, viz. parvifloron D, F, and G (1–3, respectively). Parvifloron G was isolated for the first time from P. ecklonii. The in vitro bio-evaluation of the methanolic extract of P. ecklonii and its isolated compounds against alpha-glucosidase showed that 3 exhibited moderate inhibitory activity with IC50 values of 41.3 ± 1.2 μg/mL. Molecular docking analysis confirmed the alpha-glucosidase inhibitory activity demonstrated by 3. Additionally, strong antioxidant capacities were demonstrated by 3 and 1 on ORAC (28726.1 ± 8.1; 3942.9.6.6 ± 0.1 µM TE/g), respectively, which were comparable with the reference antioxidant epigallocatechingallate (EGCG). Furthermore, 3 also showed strong activity on TEAC (3526.1 ± 0.6 µM TE/g), followed by 2 (1069.3 ± 2.4 µM TE/g), as well as on FRAP (1455.4 ± 2.0 µM AAE/g). The methanolic extract of P. ecklonii is a rich source of abietane diterpenes with strong antioxidant activities. This is the first scientific report on alpha-glucosidase and alpha-amylase inhibitory activities, molecular docking, and antioxidant capacities of P. ecklonii constituents.

The Physiology of Insulin Clearance.

In the 1950’s, Dr. I. Arthur Mirsky first recognized the possible importance of insulin degradation changes to the pathogenesis of type 2 diabetes. While this mechanism was ignored for decades, insulin degradation is now being recognized as a possible factor in diabetes risk. After Mirsky, the relative importance of defects in insulin release and insulin resistance were recognized as risk factors. The hyperbolic relationship between secretion and sensitivity was introduced, as was the relationship between them, as expressed as the disposition index (DI). The DI was shown to be affected by environmental and genetic factors, and it was shown to be differentiated among ethnic groups. However, the importance of differences in insulin degradation (clearance) on the disposition index relationship remains to be clarified. Direct measure of insulin clearance revealed it to be highly variable among even normal individuals, and to be affected by fat feeding and other physiologic factors. Insulin clearance is relatively lower in ethnic groups at high risk for diabetes such as African Americans and Hispanic Americans, compared to European Americans. These differences exist even for young children. Two possible mechanisms have been proposed for the importance of insulin clearance for diabetes risk: in one concept, insulin resistance per se leads to reduced clearance and diabetes risk. In a second and new concept, reduced degradation is a primary factor leading to diabetes risk, such that lower clearance (resulting from genetics or environment) leads to systemic hyperinsulinemia, insulin resistance, and beta-cell stress. Recent data by Chang and colleagues appear to support this latter hypothesis in Native Americans. The importance of insulin clearance as a risk factor for metabolic disease is becoming recognized and may be treatable.

Inflammation at Site of Insulin Infusion Diminishes Glycemic Control

The approximation of euglycemia is the most effective means of preventing diabetic complications, which is achieved through effective insulin delivery. Recent reports indicate that insulin phenolic preservatives, which are found in all commercial insulin formulations, are cytotoxic, pro-inflammatory and induce secondary fibrosis. Therefore, we hypothesize that these preservatives induce an inflammatory response at the site of insulin infusion leading to diminished glycemic control and adverse pharmacokinetic outcomes. Insulin degradation by inflammatory cell proteases was quantitated following protease treatment in vitro. A modified murine air pouch model was utilized to evaluate the relative inflammatory responses following infusions of saline, insulin preservatives, and insulin, utilizing the adjuvant irritant thioglycolate. Blood glucose levels were monitored in diabetic mice with and without air pouch irritation. A pharmacokinetic analysis evaluated insulin effectiveness for diabetic mice between these two conditions.Inflammatory cells are significantly present in insulin preservative-induced inflammation, which effects diminished blood glucose control by both insulin uptake and degradation. Insulin containing these preservatives resulted in similar degrees of inflammation as observed with the irritant thioglycolate. These studies imply that the preservative agents found in commercial insulin formulations induce an intense localized inflammatory reaction. This inflammatory reaction may be responsible for the premature failure of insulin infusion devices. Future studies directed at reducing this inflammatory reaction may prove to be an important step in extending the lifespan of insulin infusion devices.

Effects of some anti-diabetic herbal extracts on the insulin-degrading enzyme in human colon cancer Caco-2 cell line.

Objective:Type 2 diabetes mellitus (T2DM) is a condition characterized by insufficient insulin production or insulin resistance. The insulin-degrading enzyme (IDE) is responsible for degrading insulin and is a potential drug target for T2DM treatment. Numerous activities have been proposed for plant extracts, but research on the effects of plant extracts on IDE expression and activity is riddled with drawbacks.Materials and Methods:We investigated the effect of Phaseolus vulgaris, Allium cepa, Portulaca oleracea, Cinnamomum verum, and Citrullus colocynthis extracts on the expression and activity of IDE in the Caco-2 cell line.Results: Findings of RT-PCR showed that IDE gene expression was reduced following treatment with P. vulgaris, C. colocynthis, and C. verum extracts. The results of IDE activity with fluorogenic peptide substrate V also indicated that P. vulgaris, C. colocynthis, and P. oleracea extracts reduced IDE activity in a significant and dose-dependent manner.Conclusion:The hydroalcoholic extracts studied, except for A. cepa, can prevent insulin degradation by reducing the expression and activity of the IDE enzyme. This new insight into the effects of herbal medicines on IDE activity can help future studies.

IL-22 and its interaction with amino acid and glycolipid metabolite in polycystic ovary syndrome (PCOS) patients

IL-22 and its interaction with amino acid and glycolipid metabolite in polycystic ovary syndrome (PCOS) patients

A 3-year-old boy with congenital absence of the portal vein presenting fasting hypoglycemia and postprandial hyperglycemia with hyperinsulinemia

A 3-year-old boy developed hypoglycemia with convulsions, rectal bleeding and hyperammonemia. Three-dimensional computed tomography scanning revealed the missing main branch of the portal vein, which confirmed the diagnosis of congenital absence of the portal vein (CAPV). Oral glucose tolerance test showed hyperglycemia with hyperinsulinemia at 30 min, whereas hypoglycemia was observed during fasting and at 120 min. Postprandial hyperglycemia was observed repeatedly. Repetitive hypoglycemia and rectal bleeding were uncontrollable. Living-donor liver transplantation, which was successfully performed, was apparently effective in preventing fasting hypoglycemia and improving hyperammonemia; rectal bleeding ceased after the surgery. Considerably, CAPV could result in hyperinsulinemia with partial failure of hepatic insulin degradation and hypoglycemia due to the lack of glycogen accumulation. Postprandial hyperglycemia could result from the fact that glucose-abundant splanchnic blood directly flows into the systemic circulation through the portosystemic shunt. Therefore, it is important to be aware of CAPV as a cause of glucose metabolism abnormalities including fasting hypoglycemia and postprandial hyperglycemia with hyperinsulinemia.

The manifold roles of protein S-nitrosylation in the life of insulin.

Insulin, which is released by pancreatic islet β-cells in response to elevated levels of glucose in the blood, is a critical regulator of metabolism. Insulin triggers the uptake of glucose and fatty acids into the liver, adipose tissue and muscle, and promotes the storage of these nutrients in the form of glycogen and lipids. Dysregulation of insulin synthesis, secretion, transport, degradation or signal transduction all cause failure to take up and store nutrients, resulting in type 1 diabetes mellitus, type 2 diabetes mellitus and metabolic dysfunction. In this Review, we make the case that insulin signalling is intimately coupled to protein S-nitrosylation, in which nitric oxide groups are conjugated to cysteine thiols to form S-nitrosothiols, within effectors of insulin action. We discuss the role of S-nitrosylation in the life cycle of insulin, from its synthesis and secretion in pancreatic β-cells, to its signalling and degradation in target tissues. Finally, we consider how aberrant S-nitrosylation contributes to metabolic diseases, including the roles of human genetic mutations and cellular events that alter S-nitrosylation of insulin-regulating proteins. Given the growing influence of S-nitrosylation in cellular metabolism, the field of metabolic signalling could benefit from renewed focus on S-nitrosylation in type 2 diabetes mellitus and insulin-related disorders.

Study of the Degradation of Human Insulin

Study of the Degradation of Human Insulin

Differential Effects of Polyphenols on Insulin Proteolysis by the Insulin-Degrading Enzyme.

The insulin-degrading enzyme (IDE) possesses a strong ability to degrade insulin and Aβ42 that has been linked to the neurodegeneration in Alzheimer’s disease (AD). Given this, an attractive IDE-centric strategy for the development of therapeutics for AD is to boost IDE’s activity for the clearance of Aβ42 without offsetting insulin proteostasis. Recently, we showed that resveratrol enhances IDE’s activity toward Aβ42. In this work, we used a combination of chromatographic and spectroscopic techniques to investigate the effects of resveratrol on IDE’s activity toward insulin. For comparison, we also studied epigallocatechin-3-gallate (EGCG). Our results show that the two polyphenols affect the IDE-dependent degradation of insulin in different ways: EGCG inhibits IDE while resveratrol has no effect. These findings suggest that polyphenols provide a path for developing therapeutic strategies that can selectively target IDE substrate specificity.

Characterization of Enlarged Kidneys and Their Potential for Inducing Diabetes in DEK Rats.

Simple SummaryThe worldwide prevalence of diabetes mellitus (DM) in 2020 has been estimated at 463 million patients. About 90% of patients with diabetes have type 2 diabetes mellitus (T2D), caused primarily by insulin resistance and insufficiency. However, the specific etiology of T2D remains unknown. Sodium glucose cotransporter 2 (SGLT2) inhibitors are a novel class of anti-diabetic drugs that act independently of insulin and reduce blood glucose concentrations by inhibiting the reabsorption of glucose at renal proximal tubules. SGLT2 inhibitors have highlighted the role of the kidneys in glycemic control in diabetes. The kidneys have multiple roles in systemic glucose metabolism, such as glucose reabsorption, gluconeogenesis, and insulin degradation. Therefore, putative renal hyperfunction might contribute to the development of T2D. The present study characterized rats from a strain of novel type 2 diabetes model with enlarged kidneys (DEK). Their kidneys have increased parenchyma (nephrons and tubules), and uninephrectomy immediately after the onset inhibited the development of T2D for a significant period in DEK rats. These results highlight the contribution of kidney to the development of T2D, and indicate that the kidneys are therapeutic targets to prevent T2D.The kidneys participate in the regulation of systemic glucose metabolism via gluconeogenesis, insulin degradation, and the tubular reabsorption of glucose. The present study characterized rats from a strain of a novel type 2 diabetes model with enlarged kidneys (DEK). Histological and biochemical analyses of DEK rats were performed to assess the relationships between their kidneys and hyperglycemia. The kidney weight of diabetic DEK (DEK-DM) gradually increased over time from the onset of diabetes, with the glomerular number being higher in DEK-DM than in normal DEK (DEK-cont). A positive correlation between blood glucose level and kidney weight was observed in DEK-DM. The similar glomerular size and single glomerular creatinine clearance in DEK-cont and DEK-DM indicated that glomerular hypertrophy and hyperfiltration were not involved in the renal enlargement. Uninephrectomy (1/2Nx) in DEK-DM resulted in a reduction in blood glucose level at 7–28 post-operation days, with this concentration remaining lower than in Sham group until 84 days post-operation. 1/2Nx also improved systemic conditions, including reduced body weight gain, polyuria, polydipsia, and hyperphagia. Plasma concentrations of Na, total cholesterol, albumin, and total protein were higher, and urinary excretion of glucose, urea nitrogen, and proteins were lower, in the 1/2Nx than in the Sham group. Remnant kidney weight was two-fold higher in the 1/2Nx than in the Sham group 84 days later. In addition, 1/2Nx resulted in renal tubular dilatation but not in the progression of fibrosis or glomerular lesions. Taken together, these findings indicate that enlarged kidneys were associated with the onset of diabetes and with the resistance to diabetic nephropathy in DEK-DM.

Inpatient Hypoglycemia in Patients With Chronic Kidney Disease

Background: Inpatient hypoglycemia has been shown to be associated with increased mortality, more complications, and greater length of stay [1]. Patients with chronic kidney disease (CKD) and end-stage renal disease (ESRD) are at greater risks for hypoglycemia due to their decreased degradation of insulin, reduced glycogen stores, and decreased renal gluconeogenesis. With the association between inpatient hypoglycemia and increased morbidity and mortality, it is valuable to determine other risks factors that may contribute to hypoglycemia in the hospital setting. Objective: Determine the rates of hypoglycemia (glucose <70 mg/dl) and severe hypoglycemia (glucose <54 mg/dl) along with risk factors, such as type of insulin used and initial hyperglycemia at admission, for patients with diabetes mellitus (DM) and CKD/ESRD. Methods: A retrospective cohort analysis was conducted on 74,266 hospitalized patients who had DM and CKD/ESRD or DM alone, within 155 HCA Healthcare hospitals from January 2019 through June 2019. Results: Among hospitalized patients with DM, hypoglycemia was more common in patients with ESRD (n=5234) compared to patients with CKD without ESRD (n=18659) and patients without CKD/ESRD (n=52373) (34.37% vs. 23.66% vs. 12.91%, respectively, p<0.01). Similarly, severe hypoglycemia was more common in patients with ESRD compared to patients with CKD without ESRD, and patients without CKD/ESRD (18.09% vs. 11.19% vs. 5.28%, respectively, p<0.01).When evaluating patients with ESRD, a higher point of care (POC) glucose at time of admission was associated with an increased risk of subsequent hypoglycemia. Within the first 24 hours of hospital admission, hypoglycemia was more common if the patient’s initial POC glucose was greater than 300 with an incidence of 13.67% compared to 11.21% (n=955, p=0.033). Among patients with ESRD and an admission glucose > 300, having an order for dialysis within the first 24 hours of admission was associated with increased risk of hypoglycemia (OR= 1.2181, p=0.0014). Diabetics with subsequent hypoglycemia had higher initial glucose levels on admission when compared to diabetics who did not experience hypoglycemia during their hospital stay. Mean = 221.61±82.32 vs 205.54±74.51 (p=0.000002). Conclusion: In hospitalized patients with DM, CKD and ESRD are associated with increased risk of hypoglycemia. Amongst diabetics, patients with ESRD and CKD account for over 50% of cases of severe hypoglycemia. Severe hypoglycemia occurred in 18% of patients who had both diabetes and ESRD. An elevated glucose at admission is associated with a subsequent hypoglycemia in patients with ESRD. It is important to manage DM in patients with CKD and ESRD carefully as they may be more likely to experience hypoglycemia due to overcorrection and decreased clearance of insulin.1.Hulkower et al., Diabetes Manag 2014 Mar;4(2):165–176.

Hydroxypyridinethione Inhibitors of Human Insulin-Degrading Enzyme.

Insulin-degrading enzyme (IDE) is a human mononuclear Zn2+ -dependent metalloenzyme that is widely regarded as the primary peptidase responsible for insulin degradation. Despite its name, IDE is also critically involved in the hydrolysis of several other disparate peptide hormones, including glucagon, amylin, and the amyloid β-protein. As such, the study of IDE inhibition is highly relevant to deciphering the role of IDE in conditions such as type-2 diabetes mellitus and Alzheimer disease. There have been few reported IDE inhibitors, and of these, inhibitors that directly target the active-site Zn2+ ion have yet to be fully explored. In an effort to discover new, zinc-targeting inhibitors of IDE, a library of ∼350 metal-binding pharmacophores was screened against IDE, resulting in the identification of 1-hydroxypyridine-2-thione (1,2-HOPTO) as an effective Zn2+ -binding scaffold. Screening a focused library of HOPTO compounds identified 3-sulfonamide derivatives of 1,2-HOPTO as inhibitors of IDE (Ki values of ∼50 μM). Further structure-activity relationship studies yielded several thiophene-sulfonamide HOPTO derivatives with good, broad-spectrum activity against IDE that have the potential to be useful pharmacological tools for future studies of IDE.

Targeting Insulin-Degrading Enzyme in Insulin Clearance.

Hepatic insulin clearance, a physiological process that in response to nutritional cues clears ~50–80% of circulating insulin, is emerging as an important factor in our understanding of the pathogenesis of type 2 diabetes mellitus (T2DM). Insulin-degrading enzyme (IDE) is a highly conserved Zn2+-metalloprotease that degrades insulin and several other intermediate-size peptides. Both, insulin clearance and IDE activity are reduced in diabetic patients, albeit the cause-effect relationship in humans remains unproven. Because historically IDE has been proposed as the main enzyme involved in insulin degradation, efforts in the development of IDE inhibitors as therapeutics in diabetic patients has attracted attention during the last decades. In this review, we retrace the path from Mirsky’s seminal discovery of IDE to the present, highlighting the pros and cons of the development of IDE inhibitors as a pharmacological approach to treating diabetic patients.

Reduction of enzymatic degradation of insulin via encapsulation in a lipidic bicontinuous cubic phase

Oral delivery of the protein drug insulin is not currently possible due to rapid degradation of the secondary structure in low pH conditions in the stomach and under the influence of digestive enzymes in the gastrointestinal tract. Effective oral delivery of insulin and other protein- or peptide-based drugs will, therefore, require encapsulation in a material or nanoparticle. Herein we investigate the ability of the lipid bicontinuous cubic phase formed by two lipids, monoolein (MO) and phytantriol (PT), to protect encapsulated insulin from degradation by the enzyme chymotrypsin, typically found in the small intestine. High encapsulation efficiency (>80%) was achieved in both lipid cubic phases with retention of the underlying cubic nanostructure. Release of insulin from the cubic matrix was shown to be diffusion-controlled; the release rate was dependent on the cubic nanostructure and consistent with measured diffusion coefficients for encapsulated insulin. Encapsulation was shown to significantly retard enzymatic degradation relative to that in water, with the protective effect lasting up to 2 h, exemplifying the potential of these materials to protect the encapsulated protein payload during oral delivery.

Role of Wnt signaling pathways in type 2 diabetes mellitus.

Type 2 diabetes mellitus (T2DM) has become a major global public health issue in the twenty-first century and its incidence has increased each year. Wnt signaling pathways are a set of multi-downstream signaling pathways activated by the binding of Wnt ligands to membrane protein receptors. Wnt signaling pathways regulate protein expression and play important roles in protecting the body's normal physiological metabolism. This review describes Wnt signaling pathways, and then aims to reveal how Wnt signaling pathways participate in the occurrence and development of T2DM. We found that Wnt/c-Jun N-terminal kinase signaling was closely associated with insulin resistance, inflammatory response, and pancreatic β-cell and endothelial dysfunction. β-catenin/transcription factor 7-like 2 (TCF7L2)-mediated and calcineurin/nuclear factor of activated T cells-mediated target genes were involved in insulin synthesis and secretion, insulin degradation, pancreatic β-cell growth and regeneration, and functional application of pancreatic β-cells. In addition, polymorphisms in the TCF7L2 gene could increase risk of T2DM according to previous and the most current results, and the T allele of its variants was a more adverse factor for abnormal pancreatic β-cell function and impaired glucose tolerance in patients with T2DM. These findings indicate a strong correlation between Wnt signaling pathways and T2DM, particularly in terms of pancreatic islet dysfunction and insulin resistance, and new therapeutic targets for T2DM may be identified.

The Transition Pathway Between the Closed and Open States of Insulin Degrading Enzyme Shows Two Distinct Motions: Rotation Around a Hinge and Grinding Between Two Domains

Insulin Degrading Enzyme (IDE) is involved in the degradation of insulin, amylin, and glucagon, peptides key in controlling blood glucose level. IDE has two ∼55 kDa homologous N- and C-terminal domains (IDE-N and IDE-C), each containing two homologous subdomains (D1 and D2 for IDE-N and D3 and D4 for IDE-C). For IDE to degrade its substrates, both IDE-N and IDE-C must come together for the catalytic cleft to assume a closed conformation. To investigate the mechanism through which IDE transitions between its closed and open conformations, we combined closed state crystal structures with open state CryoEM structures to run molecular dynamics simulations following the string method with swarms of trajectories and bias-exchange umbrella sampling protocols. Our analysis shows that IDE undergoes two distinct modes of motion when opening: a rotation about the hinge joining IDE-N to IDE-C (termed the hinge motion), and a rotation about the dihedral angle between the plane containing D1, D2, and D3 and the plane containing D2, D3, and D4 (termed the grinding motion). This observation matches well with multiple-body analysis performed in Relion used to deduce the protein's motion from available CryoEM data. Whereas the hinge opening motion remains relatively constant throughout the trajectory, the grinding motion appears to stop and restart. Our free energy profile constructed from MD simulations also reveals that IDE passes through a minimum energy state near its closed conformation, and our analysis suggests that this could be attributed to π-π interaction between residues W495 and F218. Our simulations and analysis provide detailed insight into the physical mechanism for the conformational transition between the closed and open states of IDE.

Insulin signaling in health and disease.

The molecular mechanisms of cellular insulin action have been the focus of much investigation since the discovery of the hormone 100 years ago. Insulin action is impaired in metabolic syndrome, a condition known as insulin resistance. The actions of the hormone are initiated by binding to its receptor on the surface of target cells. The receptor is an α2β2 heterodimer that binds to insulin with high affinity, resulting in the activation of its tyrosine kinase activity. Once activated, the receptor can phosphorylate a number of intracellular substrates that initiate discrete signaling pathways. The tyrosine phosphorylation of some substrates activates phosphatidylinositol-3-kinase (PI3K), which produces polyphosphoinositides that interact with protein kinases, leading to activation of the kinase Akt. Phosphorylation of Shc leads to activation of the Ras/MAP kinase pathway. Phosphorylation of SH2B2 and of Cbl initiates activation of G proteins such as TC10. Activation of Akt and other protein kinases produces phosphorylation of a variety of substrates, including transcription factors, GTPase-activating proteins, and other kinases that control key metabolic events. Among the cellular processes controlled by insulin are vesicle trafficking, activities of metabolic enzymes, transcriptional factors, and degradation of insulin itself. Together these complex processes are coordinated to ensure glucose homeostasis.