Article

Human recombinant insulin and amyloidosis: An unexpected association

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Abstract

A 48-year-old patient with diabetes mellitus was treated with human (recombinant) insulin. He developed cutaneous amyloidosis twice at different locations where subcutaneous insulin had been injected. There were no signs of systemic amyloidosis. Additional pathological-anatomical investigations demonstrated insulin in one (the most recent) amyloid tumour. A limited number of similar cases have been reported in the literature, although mostly associated with porcine insulin. Cutaneous amyloidosis may be associated with local injections of human (recombinant) insulin. One should therefore also consider this diagnosis when finding tumours at sites where insulin has been injected.

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... A large number of patients with diabetes are being treated with subcutaneous insulin injection [1]. Insulin allergy in patient with diabetes is a rare condition [2]. ...
... Common dermatological complications associated with subcutaneous insulin injection include specifically lipohypertrophy and lipoatrophy [1,8,13]. Other cutaneous complications may be associated with repeated subcutaneous injections [1,8,15,16]. ...
... Common dermatological complications associated with subcutaneous insulin injection include specifically lipohypertrophy and lipoatrophy [1,8,13]. Other cutaneous complications may be associated with repeated subcutaneous injections [1,8,15,16]. After repeated use, the needle can become significantly deformed, and this often causes more pain and increases contaminations at injection site [17]. ...
... Diabetes mellitus is a highly prevalent illness and a number of diabetic patients require treatment with subcutaneous insulin injections. A previous study demonstrated that insulin injections are associated with local amyloidosis (9). Usually, these are case reports of patients who have detected an abnormal mass in the injection site (10)(11)(12). ...
... The masses formed upon amyloid injection were waxy bodies of a white-yellow color and a size of ~10x10x2 mm 3 . The masses appeared similar to the areas of lipohypertrophy observed in human diabetic cases as previously reported (9,25). ...
Article
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Localized deposits of amyloid structures are observed in various pathological conditions. One example of when local amyloidosis occurs is following repeated insulin injections in diabetic patients. The present study aimed to simulate the same condition in mice. To obtain the amyloid structures, regular insulin was incubated at 57°C for 24 h. The subsequently formed amyloid fibrils were analyzed using the Congo red absorbance test, as well as transmission electron microscopy images, and then injected into mice once per day for 21 consecutive days. Firm waxy masses were developed following this period, which were excised, prepared as thin sections and stained with hematoxylin and eosin, Congo red and Sudan black. Histological examination revealed that these masses contained adipose cells and connective tissue, in which amyloid deposition was visible. Thus, localized amyloidosis was obtained by the subcutaneous injection of insulin fibrils. The present results may be of further use in the development of models of amyloid tumors.
... In this respect it is interesting to mention AIns (insulin) amyloidosis. This rare localized type of nodular amyloidosis is an iatrogenic type of amyloid in which insulin-derived amyloid is deposited in abdominal fat tissue after long periods of repeated insulin injections in that particular site [ 36,37 ] . A constant feature of this type of amyloid seems to be a negative immunohistochemical staining of serum amyloid P component (SAP) [ 37 ] . ...
Chapter
Aspiration of abdominal subcutaneous fat tissue is a safe, inexpensive, reliable, minimally invasive, and easy procedure with high diagnostic yield that can be done early after clinical suspicion of amyloidosis and can be repeated frequently during the course of the disease. Material obtained in this way is highly suitable for detection, typing, and quantification of amyloid and can also be used for chemical tissue analysis using proteomic techniques. The procedure has great potential to enable dynamic research of local tissue factors involved both in deposition and in removal of amyloid in vivo. In the Appendix, we describe the current practice of this procedure in our center.
... Previous studies with model membranes had demonstrated the crucial role played by the associated biomembranes in affecting the aggre-J o u r n a l P r e -p r o o f Journal Pre-proof gation propensity of the molecule, thus, promoting the rapid amyloidogenesis at the point of administration. 21,22,56 Thus, protein-lipid interface plays a pivotal role in modulating insulin amyloidogenesis in vivo, similar to other well-studied amyloid proteins. Hence, it was essential to check if KR7CC could serve as an effective inhibitor molecule even at the lipid-protein interface. ...
Article
Amyloidogenic disorders are currently rising as a global health issue, prompting more and more studies dedicated to the development of effective targeted therapeutics. The innate affinity of these amyloidogenic proteins towards the biomembranes adds further complexities to the systems. Our previous studies have shown that biologically active peptides can effectively target amyloidogenesis serving as an efficient therapeutic alternative in several amyloidogenic disorders. The structural uniqueness of the PWWP motif in the de novo designed heptapeptide, KR7 (KPWWPRR-NH2) was demonstrated to target insulin fiber elongation specifically. By working on Insulin, an important model system in amyloidogenic studies, we gained several mechanistic insights into the inhibitory actions at the protein-peptide interface. Here, we report a second-generation non-toxic and serum stable cyclic peptide, based primarily on the PWWP motif that resulted in complete inhibition of insulin fibrillation both in the presence and absence of the model membranes. Using both low- and high-resolution spectroscopic techniques, we could delineate the specific mechanism of inhibition, at atomistic resolution. Our studies put forward an effective therapeutic intervention that redirects the default aggregation kinetics towards off-pathway fibrillation. Based on the promising results, this novel cyclic peptide can be considered an excellent lead to design pharmaceutical molecules against amyloidogenesis.
... A common mode of administration in the regular insulin and the insulin analog formulations treatment is MDSIs. However, repeated subcutaneous injections of the regular insulin and insulin analog formulations at the same site are known to cause insulin-derived amyloidosis (IDA) in clinical practice [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15]. IDA leads to a subcutaneous mass, which, because of its shape, is called an "insulin ball" [2]. ...
Article
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Background: Multiple daily subcutaneous injections (MDSIs) are mainly used for formulating an insulin therapy for diabetic patients; however, they also cause insulin-derived amyloidosis (IDA) and lead to poor glycemic control. In addition, for the continuous subcutaneous insulin infusion system (CSII), precipitation frequently causes catheter occlusion and, if the precipitate in the formulations is amyloid, the injection of the insoluble amyloid into the subcutaneous tissue leads to IDA. The aim of this study was to conduct in vitro experiments and present a situation where insulin formulations cause precipitation and amyloid formation. Methods: Humulin®R and NovoRapid® were used as model formulations for MDSIs and CSII, respectively. The generation of the precipitation was evaluated by measuring turbidity, and amyloid formation was evaluated by using Thioflavin T. Humulin®R was mixed with saline buffer solutions and glucose solutions to evaluate the effect of dilution. In addition, we created an experimental system to consider the effect of the time course of condition changes, and investigated the effects of insulin concentration, m-cresol existence, and pH change on the generation of the precipitate and amyloid in the formulation. Results: In both the original and diluted formulations, physical stimulation resulted in the formation of a precipitate, which in most cases was an amyloid. The amyloid was likely to be formed at a near neutral pH. On the contrary, although a precipitate formed when the pH was decreased to near the isoelectric point, this precipitate was not an amyloid. Further decreases in pH resulted in the formation of amyloids, suggesting that both the positive and negative charged states of insulin tended to form amyloids. The formulation additive m-cresol suppressed amyloid formation. When additives were removed from the formulation, the amyloid-containing gel was formed in the field of substance exchange. Conclusions: To consider changes in conditions that may occur for insulin formulations, the relationship between the formation of precipitates and amyloids was demonstrated in vitro by using insulin formulations. From the in vitro study, m-cresol was shown to have an inhibitory effect on amyloid formation.
... Upon coordination with divalent zinc ions, insulin forms a hexamer, which stabilizes the insulin structure but delays the onset of activity of insulin in human body [13]. It is worth mentioning that the monomeric form of insulin is the biologically active [14] form and is prone to amyloidosis [15], and it occurs at the site of injection [16][17][18][19][20], which not only complicates the DM management but also interferes with the diagnosis of the systemic amyloidosis [21]. Hence, a stabilizer that can reduce the insulin aggregation propensities will certainly have clinical application [22]. ...
Article
Subcutaneous insulin delivery serves as the major treatment for the ever-increasing spread of type II diabetes worldwide. However, long-term exposure to insulin results in local aggregates at the site of injection. This therapeutic concern accentuates the need to develop newer effective excipients to stabilize the insulin in pharmaceutical formulations. The fact that in normal physiological conditions, insulin interacts with the amylin hormone co-secreted from the pancreas, we targeted a peptide-mimetic approach based on the amylin sequence. The amylin-fibrillating core (NL6- N²²FGAIL²⁷ from the human Islet Amyloid Poly-Peptide) and its derivative NFGAXL (NL6X, X = 2-aminobenzoic acid) were used as potential inhibitory peptides against insulin amyloidogenesis. The fibrillation kinetics in the presence of the inhibitors was studied using an array of biophysical and microscopic techniques. High-resolution NMR spectroscopy enabled probing of the inhibitory interaction at an atomic resolution. Our results highlight the potential of using the naturally evolved NL6 peptide as an effective inhibitor against insulin fibrillation.
... The amyloid fibril protein is derived from the injected insulin itself and forms a hard subcutaneous mass at the injection site. 1 Several case reports have been published, including ours, 2 describing a total of 11 patients with amyloid masses that were immunoreactive to insulin antibodies at the insulin injection sites. [1][2][3][4][5][6][7][8] In a previous report, we described a patient in whom this form of local amyloidosis, also known as "insulin ball", caused poor glycemic control and an increase in insulin dose requirements. 2 insulin values from 0 to 120 min (AUC 0-120min ) was calculated using the trapezoidal method, and the peak insulin value (C max ) was established. ...
Article
Full-text available
Insulin-derived amyloidosis is a rare skin-related complication of insulin therapy. The purpose of this study was to show the effects of insulin-derived amyloidosis on blood glucose levels, insulin dose requirements, and insulin absorption. Seven patients were found to have insulin-derived amyloidosis in our hospital. The clinical characteristics and insulin therapy of the seven patients were investigated. Insulin absorption was studied by comparing the serum insulin levels after insulin injections into insulin-derived amyloidosis sites versus injections into normal sites in four patients. When the insulin-derived amyloidosis was discovered, the mean hemoglobin A1c level was 9.3%, and the mean daily insulin dose was 57 units. After changing the injection sites to avoid the insulin-derived amyloidosis, the blood glucose concentrations improved, and the mean daily insulin dose could be reduced to 27 units (P = 0.035; 53% reduction). The insulin absorption at insulin-derived amyloidosis sites was 34% of that at normal sites (P = 0.030). Insulin-derived amyloidosis caused poor glycemic control and increased insulin dose requirements due to impairments in insulin absorption.
... In this respect it is interesting to mention AIns (insulin) amyloidosis. This rare localized type of nodular amyloidosis is an iatrogenic type of amyloid in which insulin-derived amyloid is deposited in abdominal fat tissue after long periods of repeated insulin injections in that particular site [ 36,37 ] . A constant feature of this type of amyloid seems to be a negative immunohistochemical staining of serum amyloid P component (SAP) [ 37 ] . ...
Chapter
Aspiration of abdominal subcutaneous fat tissue is a safe, inexpensive, reliable, minimally-invasive, and easy procedure with high diagnostic yield that can be done early after clinical suspicion of amyloidosis and can be repeated frequently during the course of the disease. Material obtained in this way is highly suitable for detection, typing, and quantification of amyloid and can also be used for chemical tissue analysis using proteomic techniques. The procedure has great potential to enable dynamic research of local tissue factors involved both in deposition and in removal of amyloid in vivo. In the Appendix of this chapter we describe the current practice of this procedure in our center.
... In vivo, insulin amyloid fibrils have been observed in diabetic patients as part of cutaneous amyloid tumors at the sites where frequent insulin injections are made, in as many various locations as shoulders, arms, thighs and abdomen (Störkel et al., 1983;Dische et al., 1988;Albert et al., 2007;Yumlu et al., 2009;Sie et al., 2010). These deposits results into poor absorption of subsequently injected insulin, leading to impaired control of hyperglycemia, and a need to increase insulin dosage (Nagase et al., 2014). ...
Article
Insulin is a therapeutic protein whose amyloid formation is reported in diabetic patients. Four anhydride compounds were used in the current study in order to experiment their potential reducing effect on insulin propensity to form amyloid fibrils. The modified forms (obtained with succinic-, 3,3-dimethylglutaric, 2-phenylglutaric-, and (2-Dodecen-1-yl) succinic anhydride), were first characterized with regard to melting temperature (Tm), changes in secondary structure percentage and hydrophobic surface. Fibril formation was then assessed by Congo red absorbance kinetics and transmission electron microscopy. Functionality was investigated with the use of an insulin tolerance test in NMRI mice. Finally, 10 ns molecular dynamics simulations were performed during which structural changes, potential energy, gyration radius, RMSD, and accessible surface area were monitored. In all cases, α-helical structure content of the modified forms was reduced, but thermal stability and structural compactness of modified insulin were increased except in case of the dodecenylated species. All modified insulin forms undergo amorphous aggregation instead of amyloid fibrils formation, and dodecenylated insulin makes the largest amorphous aggregates. In silico results were overall in accordance with in vitro studies. Finally, only succinylated insulin was functional, although dimethylglutaric-modified insulin started to show some activity after 2 h.
... Before the publication of the paper by Jaunmuktane et al. [1], Irwin et al. [6] had found "no evidence to support concerns that NDAPs underlying AD transmit disease in humans despite evidence of their cell-to-cell transmission in model systems of these disorders". A number of hormones are involved in amyloidoses: atrial natriuretic factor, found in isolated cardiac atrial amyloidosis; insulin -bovine [7], porcine [8] or even recombinant human [9] -, which form subcutaneous nodules at sites of injection; and amylin, calcitonin, GH, and prolactin (PRL), found in amyloidosis associated to insulinoma or type 2 diabetes mellitus, medullary thyroid cancer, GH-secreting and PRL-secreting pituitary adenomas, respectively [10][11][12]. The paper by Jaunmuktane et al. [1] misses literature data on intratumoral amyloidosis in various polypeptide hormone producing tumors [13], including GH-secreting and PRL-secreting adenomas [14,15]. ...
Article
Great interest has recently been focused on a paper reporting characteristic deposits of amyloid-β protein associated with Alzheimer's disease in brains of adults who died of Creutzfeldt-Jakob disease. As they had contracted such disease after treatment with prion-contaminated human growth hormone extracted from cadaver-derived pituitaries, the authors have suggested that interhuman transmission of Alzheimer's disease had occurred. Our previous research led us to find that amyloid-forming peptides share amino acid sequence homology, summarized by a motif. Here, we probed the amino acid sequence of human growth hormone for such a motif, and found that 2 segments fit the motif and are potentially amyloid-forming. This finding was confirmed by Aggrescan, another well-known software for the prediction of amyloidogenic peptides. Our results, taken together with data from the literature that are missing in the aforementioned paper and associated commentaries, minimize the contagious nature of the iatrogenically-acquired coexistence of Creutzfeldt-Jakob disease and Alzheimer's disease. In particular, the above mentioned paper misses literature data on intratumoral amyloidosis in growth hormone- and prolactin-secreting adenomas, tumors relatively frequent in adults, which are often silent. It cannot be excluded that some pituitaries used to extract growth hormone contained clinically silent microadenomas, a fraction of which containing amyloid deposits, and patients might had received a fraction of growth hormone (with or without prolactin) that already was an amyloid seed. The intrinsic amyloidogenicity of growth hormone, in the presence of contaminating prion protein (and perhaps prolactin as well) and amyloid-β contained in some cadavers' pituitaries, may have led to the observed co-occurring of Creutzfeldt-Jakob disease and Alzheimer's disease. © Georg Thieme Verlag KG Stuttgart · New York.
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Localized insulin-derived amyloid masses occasionally form at the site of repeated insulin injections in patients with insulin-dependent diabetes and cause subcutaneous insulin resistance. Various kinds of insulin including porcine insulin, human insulin, and insulin analogues reportedly formed amyloid fibrils in vitro and in vivo, but the impact of the amino acid replacement in insulin molecules on amyloidogenicity is largely unknown. In the present study, we demonstrated the difference in amyloid fibril formation kinetics of human insulin and insulin analogues, which suggests an important role of the C-terminal domain of the insulin B chain in nuclear formation of amyloid fibrils. Furthermore, we determined that cyclodextrins, which are widely used as drug carriers in the pharmaceutical field, had an inhibitory effect on the nuclear formation of insulin amyloid fibrils. These findings have significant implications for the mechanism underlying insulin amyloid fibril formation and for developing optimal additives to prevent this subcutaneous adverse effect.
Article
A 59-year-old patient with diabetes mellitus had been treated with human recombinant insulin for 4 years. He developed a solid mass on his left abdomen at the insulin injection site, which had an overlying pigmented verrucous plaque and keratinized papules, similar to acanthosis nigricans (AN). On histological examination, the mass was found to contain a deposit of amyloid in the dermis, with hyperkeratosis, papillomatosis and acanthosis in the epidermis. Using immunohistochemistry, the amyloid deposits were found to be positive for insulin. A few cases of localized insulin-derived amyloid deposits at injection sites have been reported previously, but none had significant epidermal changes. The coexistence of dermal insulin-derived amyloidosis and an overlying AN-like change, as found in our patient, has not been reported previously, to our knowledge. The presence of a tumour-like lesion at the injection site should be carefully examined, as injection of insulin into amyloid deposits can result in insulin resistance.
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Skin is one of the important organs affected by amyloidosis which is characterized by extracellular deposition of fibrillary proteins having homogenous, eosinophilic on routine staining with distinct tinctorial properties. Nodular cutaneous amyloidosis is rare and may affect dermis, subcutis and also vascular walls. Nodular amyloid deposits in the deeper dermis occurring at the site of insulin injection are a rare observation, which is described here. This description indicates that cutaneous amyloidosis may be associated with local subcutaneous injections of insulin and may clinically mimic a neoplasm or lipodystrophic lesion.
Article
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Amyloidomas are rare tumors composed of deposits of amyloid protein not associated with systemic amyloidosis. They can present as an initial manifestation of a systemic disease process or can be a completely localized phenomenon. We present a case of amyloidoma associated with insulin injection site found incidentally in an 80-year-old male with multiple co-morbidities who presented with diverticulitis associated bleeding. A subcutaneous abdominal mass was found on physical examination. Imaging revealed a 5 cm × 1.6 cm homogenous subcutaneous lesion. A fine-needle aspiration (FNA) and core biopsy were performed under ultrasound guidance to reveal amorphous material proven to be amyloidosis at insulin injection sites (AIns) type amyloid. The patient had no treatment for this lesion and has had his care triaged to his more serious health problems. This is the first case of AIns type amyloidoma associated with insulin injection site reported in cytology literature. We highlight the cytologic findings and diagnostic pitfalls. As the incidence of diabetes is increasing, cytopathologists may encounter this lesion more often on FNA.
Article
The present study was carried out to clarify the histology, imaging features, and insulin absorption in 10 diabetic patients who developed subcutaneous masses in the abdomen during insulin therapy. The histological findings showed mainly amyloid depositions in 4 cases and mainly fibrosis in 6 cases. Foreign body giant cells and epithelioid cell granulomas were present in or near the amyloid depositions and the fibrosis. We also found phagocytosis of insulin by histiocytes in the granulomas. Amyloid depositions appeared as low-echoic masses with deep attenuation on body surface ultrasonography and as high density masses on computed tomography. A marked reduction of insulin absorption was recognized, not only in the cases of amyloid deposition, but also in the cases of fibrosis. We speculate that the observed fibrosis represents an earlier stage that occurs before amyloid deposition. In conclusion, in addition to an ocular inspection and palpation, it is important to perform an imaging examination in some cases for early detection of subcutaneous mass at the stage of fibrosis.
Article
The patient was a 91-year-old male who was diagnosed with type 2 diabetes at 60 years of age, and started insulin treatment at 78 years of age. His HbAlc was 10.6 % (NGSP value) at the first visit to the hospital, despite the administration of 61 units of insulin. An abdominal examination revealed elastic-hard masses at the insulin injection sites in his abdomen. The patient was admitted for further examination. Tl- and T2-weighted MRI images showed low intensity masses with no signal inhibition on fat-suppressed images. His skin biopsy samples were pale with an eosinophilic nonstructural substance stained orange with Congo-Red. The lesions were thus diagnosed as insulin injection-induced local amyloid deposition. The required insulin dose was markedly decreased after changing the insulin injection sites. In addition, the blood insulin levels were markedly lower when insulin was injected into the amyloid-deposited region than into healthy skin, thus indicating markedly reduced insulin absorption from the region containing amyloid. The first case of reduced insulin absorption due to insulin injection-induced local amyloid deposition reported as a possible cause of poor blood glucose control.
Article
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The formation of insulin amyloid can dramatically impact glycemic control in patients with diabetes, making it an important therapeutic consideration. In addition, the cost associated with the excess insulin required by patients with amyloid is estimated to be $3K per patient per year, which adds to the growing financial burden of this disease. Insulin amyloid has been observed with every mode of therapeutic insulin administration (infusion, injection and inhalation), and the number of reported cases has increased significantly since 2002. The new cases represent a much broader demographic, and include many patients who have used exclusively human insulin and human insulin analogs. The reason for the increase in case reports is unknown, but this review explores the possibility that changes in patient care, improved differential diagnosis and/or changes in insulin type and insulin delivery systems may be important factors. The goal of this review is to raise key questions that will inspire proactive measures to prevent, identify and treat insulin amyloid. Furthermore, this comprehensive examination of insulin amyloid can provide insight into important considerations for other injectable drugs that are prone to form amyloid deposits.
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We examined the feasibility of ultrasound diagnosis of insulin-derived localized amyloidosis (IDLA). In addition to ultrasound detectability and findings, the insulin absorption rate, insulin dosage and hemoglobin A1c (HbA1c) levels before and after shifting the insulin injection site were investigated for 22 cases of IDLA. The detectability of IDLA on ultrasound was 100%; 59.1% was palpable lumps and 40.9% was not palpable. The palpable type had lower echo intensity and were harder than the non-palpable type. Blood flow decreased in IDLA, especially in the palpable type. IDLA, especially the palpable type, had a low insulin absorption rate. HbA1c level and insulin dosage decreased after shifting the injection site. The palpable type had more insulin reduction than the non-palpable type. Characteristic ultrasound images of IDLA were acquired. As the non-palpable type could be identified by ultrasound, its diagnosis encourages changing the insulin injection site; hence, ultrasound diagnosis of IDLA can enhance insulin treatment.
Article
Objective The purpose of this study was to describe the imaging characteristics of subcutaneous amyloid deposits occurring at sites of insulin injection, commonly known as “insulin balls,” in diabetic patients on ultrasound, CT, and MRI with pathologic correlation. Materials and methodsWe retrospectively reviewed the radiographic findings of 14 lesions in 9 patients diagnosed with subcutaneous amyloid deposits at our institution between 2005–2015. Three board-certified radiologists analyzed the following: (1) the shape, size, margin, morphologic characteristics, and blood flow on US using the color Doppler signal, (2) shape, size, margin, attenuation, and presence or absence of contrast enhancement on CT, and (3) shape, size, margin, signal intensity, and presence or absence of contrast enhancement on MRI. ResultsAll lesions showed ill-defined hypovascular subcutaneous nodules with irregular margins. The median diameter of lesions was 50.4 mm on US, 46.8 mm on CT, and 51.4 mm on MRI. The internal echogenicity of subcutaneous amyloid deposits was hypoechoic and heterogeneous on US. All lesions showed isodensity compared to muscle with irregular margins and minimal contrast enhancement on CT. Both T1- and T2-weighted MR images showed low signal intensity compared with subcutaneous fat. Normal diffusion and minimal contrast enhancement were seen. Conclusions Subcutaneous amyloid deposits which cause insulin resistance are typically ill-defined and heterogeneous hypovascular subcutaneous nodules with irregular margins on imaging that correspond to insulin injection sites. It is also characteristic that T2WI shows low intensity compared with fat on MRI, reflective of the amyloid content.
Article
Background In diabetic patients, subcutaneous insulin injection may cause several types of injection site-related lesions, such as lipoatrophy, insulin-induced cutaneous lipohypertrophy (IICL), allergic reaction, and iatrogenic localized insulin-derived amyloidosis (LIDA). Among these complications, both IICL and LIDA present as tumor-like and slow growing lesions; and they may be confused with one another. The clinical implication and management of IICL and LIDA are different. LIDA causes poor blood glycemic controls due to inadequate absorption of the insulin. Thus, accurate diagnosis of the lesion is critical in diabetic patients. Review of literature LIDA is an extremely rare complication and often overlooked, it is managed by a surgical intervention. Whereas, IICL is a common side effect and can be managed by a non-surgical approach. Furthermore, in long-standing diabetics, patients may develop hypertrophic cardiomyopathy, proteinuria, peripheral, and autonomic neuropathy; these symptoms can be mistaken for a systemic amyloidosis. It is also necessary to distinguish LIDA from the systemic amyloidosis, which requires a more aggressive systemic therapy. LIDA should also be distinguished from primary cutaneous amyloidosis, with high risk of progression to a systemic amyloidosis. In this effort we reviewed 25 published manuscripts, including case reports and case series studies. We also summarized the literature and discussed differential diagnosis, including the approach to diagnose LIDA. Conclusion The identification of amyloid material and immunoreactivity with anti-insulin antibodies are key diagnostic features of LIDA. Although several clinical and animal studies were made in recent years, the lesion is still under-diagnosed and underreported. The clinical suspicion and knowledge of the lesion play a crucial role for the accurate diagnosis of LIDA. Surgical excision of the lesion can dramatically decrease insulin requirement and improve glycemic control.
Thesis
The folding and stability of proteins depend on the physico-chemical conditions of their environment. Especially pH, temperature, stirring and interactions with other macromolecules or with interfaces (liquid-material surfaces; air-liquid; etc.) are known to induce protein denaturation and aggregation phenomena.The control of therapeutic protein stability represents a medical and economic challenge for the pharmaceutic industry. For instance insulin, which is the most s model produced therapeutic protein, is known to form amyloid aggregates in vitro induced by hydrophobic surfaces. Amyloid aggregates are also involved in several pathologies including human and animal diseases of high economic and public health impact.This thesis focuses on amyloid aggregation at material surfaces using insulin as a model protein. Previous work from our team have demonstrated that short peptides have the ability to significantly interfere with the kinetics of surface-driven amyloid aggregation and this at sub-stoichiometric concentrations with respect to insulin. In particular peptides adopting a beta-sheet secondary structure when adsorbed on hydrophobic surfaces, were able to reduce the nucleation time of insulin aggregation.In the present work we have discovered peptide sequences presenting, again at sub-stoichiometric concentrations, two antagonistic effects on insulin aggregation kinetics. The first consists in a cooperative reduction of the nucleation time and operates via peptides bound to the material surface. The second, on the other hand, results in a powerful inhibition of both nucleation and fiber elongation via peptides remaining in solution.We have first quantitatively characterized these effects on a set of peptides presenting alternate primary sequences of the type (LK)nL, and investigated the underlying mechanisms promoting insulin nucleation. Quantitative fluorescence measurements (Thioflavin T, fluorescent labelling of the peptide) have shown that the cooperative adsorption of peptides on hydrophobic material surfaces was responsible for the reduction of the insulin nucleation time. We have then shown that the inhibitory effect results from the binding of peptides in solution to fibrillar insulin aggregates and that this effect is mediated by charges.In addition we studied the localization of the insulin nucleation and of the appearance of the first aggregates using fluorescence microscopy. We observed the preferential appearance of the first ThT positive aggregates at the solid-liquid-air triple interface undergoing high shear stress, making these regions the predominant nucleation sites.We eventually developed a technique allowing a localized and patterned growth of light-induced insulin aggregates on glass surfaces. This atypical aggregation pathway does not present any observable lag time and depends strictly on Thioflavin T. We have shown that the ThT inserted between the cross beta-sheets and which can be excited at 440 nm locally provides the energy required for the conformational transition of the native insulin into the aggregated one. This method can be used to obtain a differential amyloid growth between surface area of different hydrophobicity.
Article
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It is well known that interfaces, such as polar-nonpolar or liquid-air, play a key role in triggering protein aggregation in vitro, in particular the aggregation of peptides and proteins with the predisposition of misfolding and aggregation. Here we show that the interface present in the lungs predisposes the lungs to form aggregation of inhaled insulin. Insulin inhalers were introduced, and a large number of diabetic patients have used them. Although inhalers were safe and effective, decreases in pulmonary capacity have been reported in response to inhaled insulin. We hypothesize that the lung air-tissue interface provides a template for the aggregation of inhaled insulin. Our studies were designed to investigate the harmful potential that inhaled insulin has in pulmonary tissue in vivo, through an amyloid formation mechanism. Our data demonstrate that inhaled insulin rapidly forms amyloid in the lungs causing a significant reduction in pulmonary air flow. Our studies exemplify the importance that interfaces play in protein aggregation in vivo, illustrating the potential aggregation of inhaled proteins and the formation of amyloid deposits in the lungs. These insulin deposits resemble the amyloid structures implicated in protein misfolding disorders, such as Alzheimer's and Parkinson's diseases, and could as well be deleterious in nature.
Article
Three patients with insulin dependent type 1 diabetes mellitus and one with insulin dependent type 2 diabetes mellitus developed localized amyloid tumors at their general insulin injection sites. All 4 patients (two women and two men) were treated with modern recombinant human insulin or insulin analogues. In addition, 1 patient had used both recombinant and animal insulin. The amyloid tumors were resected and examined histologically using Congo red staining and immunohistochemistry. Insulin was found to be the major component of the amyloid tumors in all four patients. These 4 cases were diagnosed recently within a relatively short period of time, which leads to the conjecture that local insulin-derived amyloid tumors remain principally a differential diagnosis of skin tumors in insulin-dependent diabetic patients.
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A patient with Type 1 (insulin-dependent) diabetes mellitus developed localised amyloidosis at the sites of his injections of porcine insulin. A major amyloid fibril protein was extracted and, by means of its amino acid composition and amino acid sequence, it was shown to contain intact insulin molecules. Porcine insulin is the tenth protein and the first foreign protein to be chemically identified in human amyloid fibrils.
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Human and experimental amyloidosis can occur either as a generalized widespread deposit of various proteins or a localized deposit. We looked for local amyloidosis caused iatrogenically under clinical and experimental conditions. Subcutaneous tissue from one diabetic patient and six Wistar rats, which had received a continuous local infusion of 1.2 iu of insulin daily for 6 weeks, was examined histologically. In all cases the development of granulation tissue around the tip of the catheter was observed. In addition, inhomogenous extracellular deposits showing green birefringence under polarized light when stained Congo red were seen. Immunohistologically, they displayed binding of anti-insulin antibody. Electron microscopy demonstrated a typical spear-like fibrillar structure with a fibril diameter of 60 to 80 A. These findings confirmed that the deposited substance was amyloid. Iatrogenically administered protein produced in vivo amyloidosis at the site of its entry. Insulin can lead to the formation of amyloid fibrils not only in vitro but also in vivo.
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The cutaneous deposition disorders are a group of unrelated conditions characterized by the presence of either endogenous or exogenous substances within the dermis or the subcutis. Part I of this two-part series will focus on metabolic processes involved in the endogenous deposition in the various forms of amyloidosis, porphyria, colloid milium, and lipoid proteinosis. We will also review the clinical, histologic, biochemical, and ultrastructural findings relevant to each disorder. Basic mechanisms of pathogenesis, diagnostic modalities, and treatment options are also discussed.
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We present long-term follow-up data on patients with nodular localized primary cutaneous amyloidosis (NLPCA) seen at the St John's Institute of Dermatology between 1968 and 1999. This is the largest clinical follow-up study of this type of amyloid to date. Based on these cases we estimate the rate of progression of NLPCA to systemic amyloidosis to be only 7%, much lower than the 50% rate currently quoted in the literature.
Article
We describe the cytologic findings and clinical presentation of three unusual cases of amyloid tumor. Two of our patients had low-grade lymphoid malignancies and the third insulin-dependent diabetes mellitus. In no cases was amyloid suspected as the cause of mass lesion. Two of our cases presented with superficial soft tissue mass and the third with right breast masses and bilateral axillary lymph node enlargement. Air-dried slides from all aspirated cases were stained with Diff-Quik for specimen adequacy evaluation. The remaining fixed slides were stained with Papanicolaou stain. Amyloid appeared as dark-blue to purple clumps of acellular material on Diff-Quik stain, accompanied with chronic inflammatory cell infiltrates and multinucleated giant cells, simulating granulomatous inflammation. Papanicolaou stain demonstrated cyanophilic to orangophilic acellular material. Amyloid was suspected and subsequently confirmed by Congo red stain.
Article
A 59-year-old man with type 1 diabetes mellitus presented with severe resistance to subcutaneously injected insulin. Histological analysis of the injection sites, demonstrated foreign body type granulomas surrounding areas of amyloidosis. It is suggested that the granulomas were the source of an insulin-degrading enzyme (IDE) which simultaneously degraded amyloidogenic precursors into localized amyloid deposits. These findings may add insight into the role of insulin-degrading enzymes in the etiology of subcutaneous insulin resistance syndromes.