Supramolecular insulin assembly II for a sustained treatment of type 1 diabetes mellitus

Sarika Guptaa, Tandrika Chattopadhyaya, Mahendra Pal Singha, and Avadhesha Suroliaa,b,1
a National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India; and bMolecular Biophysics Unit, Indian Institute of Sciences, Bangalore 560012, India

Communicated by William J. Lennarz, State University of New York at Stony Brook, Stony Brook, NY, May 21, 2010 (received for review May 1, 2009)

protein folding hyperglycemia normoglycemia insulin-like growth factor-1

iabetes is a debilitating disease, responsible for extensive deaths worldwide (http://www.who.int/diabetes/en/). The economic impact of this is likely to be even more stupendous, because people may live for years with diabetes; their cause of death is often recorded as cardiovascular diseases, stroke, and kidney failures, all precipitating largely because of diabetes (1). Diabetes is a chronic disease characterized by either the inability of the body to produce insulin (type 1) or the failure to respond to it (type 2) (2). Attempts to replicate physiological insulin secretion, as a means of restoring the normal metabolic milieu and thereby minimizing the risk of diabetic complications, have become an essential feature of treatment of this disease. The current advocacy of intensive insulin therapy regimens involving multiple daily s.c. injections places a heavy burden of compliance on patients and has prompted interest in developing alternative, less invasive therapies. To date, attempts to exploit the nasal, oral, gastrointestinal, and transdermal routes have been mostly unsuccessful. The conventional regimen of multiple daily insulin injections for type 1 diabetes is effective in preventing postprandial blood glucose excursions, but it is limited by its inadequate control of fasting hypoglycemia (3). In the present study, we utilize the currently available knowledge of protein folding to develop a safe and long-acting supramolecular insulin assembly (SIA-I, SIA-II, and SIA-III). Insulin is a 51 residue polypeptide that exists in equilibrium in solution as a mixture of different oligomeric states. In pancreatic secretory vesicles, insulin is stored as a hexamer at physiological pH, whereas it interacts with its receptor as a monomer. Under denaturing conditions, such as low pH or in the presence of strong denaturants, insulin aggregates into amyloid fibrils. To test the hypothesis that insulin released from supramolecular insulin assembly II (SIA-II) obtained by oligomerization around physiological pH achieved better glycemic control for a sustained period of time in animal model of diabetes, SIA was developed and characterized in detail during these studies for its use as an alternative treatment to conventional insulin therapy. Insulin from two different sources (namely, bovine insulin and recombinant human insulin) were seen for their ability to form SIA-II and exert a tight glycemic
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by Th-T fluorescence. As shown in Fig. 1A, an increase in Th-T fluorescence with time was observed, which reached a maximum value at 48 h for the hormone incubated at pH 7.0, whereas for pH 2.0, Th-T fluorescence attained a maximum value at 20 h, indicating that the fibril formation is more rapid under acidic conditions. Three supramolecular insulin intermediates at pH 7.0 SIA-I, SIA-II, and SIA-III formed at 68 h, 1216 h, and 2024 h, respectively, were selected. These intermediates were further characterized structurally and release of insulin monomers from them was studied.
Release of Insulin Monomers from Its Amyloid and SIA-II Forms. To prove our hypothesis that the SIA-II form of insulin can act as a reservoir for the sustained release of the hormone for a long time, release of insulin monomers from its amyloid and various intermediates (SIA-I, SIA-II, and SIA-III) through a 8-kDa cutoff membrane was examined (Fig. S1 A and B). Fully formed amyloid fibers did not release insulin, irrespective of the pH of fibrilization. The pH 2.0 intermediates released insulin at a very slow rate, suggesting that these oligomers are sturdy and tightly associated. However, the pH 7.0 intermediate (termed SIA-II) released insulin at an appreciable rate. A linear increase in the release of insulin from SIA-II, as monitored by its absorbance at 280 nm over a period of 15 5 days, was observed (Fig. 1B). These observations were also corroborated by tyrosine fluorescence, which increased in the dialysate with time. Therefore, SIA-II was chosen so as to achieve a constant in vivo release, for maintaining basal insulin levels in the body. SIA-II intermediates were further characterized using Congored (CR) binding and FTIR, and its morphology was assessed by atomic force microscopy (AFM) and transmission electron microscopy (TEM). Congo-Red Binding. CR binds specifically to the -sheet rich struc-

tures of amyloid and has been used routinely for their detection. Both bovine and rH SIA-II exhibited weak binding to CR (Fig. 1 C and D), whereas fully grown fibers at both pH 2.0 and 7.0
Author contributions: S.G. and A.S. designed research; S.G., T.C., and M.P.S. performed research; S.G., T.C., M.P.S., and A.S. analyzed data; and S.G., T.C., and A.S. wrote the paper. The authors declare no conflict of interest.
1

To whom correspondence should be addressed. E-mail: surolia@nii.res.in.

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Diabetes is a chronic disease requiring continuous medical supervision and patient education to prevent acute secondary complications. In this study, we have harnessed the inherent property of insulin to aggregate into an oligomeric intermediate on the pathway to amyloid formation, to generate a form that exhibits controlled and sustained release for extended periods. Administration of a single dose of the insulin oligomer, defined here as the supramolecular insulin assembly II (SIA-II), to experimental animals rendered diabetic by streptozotocin or alloxan, released the hormone capable of maintaining physiologic glucose levels for >120 days for bovine and >140 days for recombinant human insulin without fasting hypoglycemia. Moreover, the novel SIA-II described here not only improved the glycemic control, but also reduced the extent of secondary diabetic complications.

control in various diabetic models, such as rat, mice, and rabbit. (Mice and rabbit data are given in SI Text). A single dose of insulin SIA-II when administered s.c. or i.m. was able to maintain a basal level of insulin in streptozotocin (STZ)induced diabetic rat for a prolonged period of >120 and >140 days for bovine and recombinant human (rH) insulin, respectively, simultaneously keeping a tight glycemic control, thus affording a long-lasting treatment against diabetes mellitus type 1 (DM-1) in experimental animals. Results
Monitoring of Insulin Fibril Formation by Thioflavin T (Th-T) Fluorescence. Fibril formation by bovine and rH insulin was monitored

Fig. 1. Formation of fibrils by insulin at 37 C (A) Kinetics of fibril formation at pH 7.0 and 2.0, monitored with 50 M Th-T for both bovine and rH insulin. (B) In vitro release of insulin from SIA-II (bovine and rH) intermediate monitored by absorbance (A) at 280 nm and intrinsic tyrosine fluorescence (F). The Th-T intensity of solution inside the dialysis membrane at 0 h and 15 days is also given. (C and D) Congo-red binding studies with native insulin, SIA-I, SIA-II, SIA-III, and amyloid insulin (rH and bovine). (E) Morphology of SIA-II intermediates and insulin fibrils using ATM: (i) insulin monomer, (ii and v) SIA-I, (iii and vi) SIA-II, (iv and vii) SIA-III intermediate, (viii) fully grown fibers at pH 7.0, 37 C, (ix) SIA-II intermediate formed at 6 h, pH 2.0, and (x) amyloid fibril formed at 20 h, pH 2.0, 37 C. (F) Twenty percent Coomassie stained gel showing a cleavage pattern of (i) rH insulin, (ii) SIA-I (68 h), (iii) SIA-II (1216 h), and (iv) SIA-III (26 h) with different dilutions of 2 mgmL proteinase K (lane 1, marker; lane 2, no proteinase K; lane 3, 11;000; lane 4, 12;000, and lane 5, 15;000). Results are mean SD of five different experiments performed in triplicate. B, bovine insulin; rH, recombinant human insulin.

showed significant binding. Thus SIA-II is a nonfibrillar structure and is devoid of amyloid-like characteristics.
Fourier Transform Infrared Spectroscopy. SIA-I, SIA-II, and SIA-III were also characterized using attenuated total reflection (ATR)FTIR. Distinct spectra corresponding to each stage were observed (Fig. S1C). SIA-II has a sharp peak at 1;647 cm1 and 1;645 cm1 for bovine and rH insulin, respectively. In contrast, the fully formed amyloid peaks at 1;630 cm1 and 1;628 cm1 for bovine and rH insulins, respectively. The FTIR spectra is in good agreement with the CR binding data showing that the conformation of SIA-II is still largely helical, albeit with an increase in the content of random coil structure. Atomic Force Microscopy. Morphology of fibers formed at pH 2.0 and 7.0 were assessed by AFM. Native insulins (both bovine and rH) at pH 7.0 and pH 2.0 show random distribution with a height of 1.3 0.21 nm, which correlates with the dimension of insulin monomers (1.11 nm) and dimers (1.49 nm) [Fig. 1E(i)]. Intermediates of the fibrilization process at pH 7.0 are shown in Fig. 1E(iiiv) for bovine insulin and Fig. 1E(vvii) for rH insulin. SIA-I represents elongated clusters having a pearl-like arrangement. In between, there are some elongated linear particles with 12 2 nm height, suggesting further association into higher oligomeric states. The SIA-II intermediate of bovine insulin is seen as a linear association of the above-mentioned elongated clusters; however, in case of rH insulin, large elongated bead-like structures with 34 10 nm height were seen [Fig. 1E(iii and vi)]. SIA-III (stage succeeding SIA-II) showed an increase in density of higher oligomeric structures. Insulin amyloids are seen as fully grown fibers at pH 7.0 [Fig. 1E(viii)]. Intermediates of the pH 2.0 fibrilization process at 67 h reveal a lateral association of two fibrils of height 7.28.3 nm, and after 20 h, large twisted fibers of 1012 nm width were observed [Fig. 1E(ixx)]. TEM studies of the intermediates were performed to see the presence of other possible assemblies and are shown in Fig. S1D. Protease Cleavage. Proteolysis experiments were performed to explore the resistance of SIA-II and SIA-III to cleavage by proteinase K. rH insulin and SIA-I were highly susceptible to cleavage by proteinase K as compared to SIA-II and SIA-III [Fig. 1F(iiv)].
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This demonstrates that both SIA-II and SIA-III adopt a higher-order oligomeric form, resistant to protease action.
In Vivo Studies in STZ-Induced DM-1 Rat Model; Treatment with SIA-II Form of Insulin. Optimization of bovine and rH insulin dosage was

done using 28 unitskg body weight, which has been reported to maintain normoglycemia in diabetic rats. When administered once or twice daily, 4 unitskg body weight was found to be ideal and did not lead to sudden fasting (preprandial) and nonfasting (postprandial) hypoglycemia. STZ-induced diabetic rats were divided into five groups, and the sixth group consisted of nondiabetic animals. Group I diabetic animals were treated with insulin (4 unitskg, i.p. daily), group IIinsulin amyloid, group III SIA-II (s.c.), group IVSIA-II (i.m.), and group VPBS-treated diabetic control rats. Insulin amyloid had no beneficial effect on the glycemic status of diabetic rats (Fig. S2A), due to negligible release of insulin monomers from the tightly packed fibrils. However, SIA-II was effective in lowering blood glucose levels. To standardize the dosage 50, 100, 200, and 400 g of the hormone oligomer were injected either s.c. or i.m. in STZ-diabetic rats. A single dose of 50 and 100 g of SIA-II maintained near-normoglycemic levels up to 10 and 30 days for bovine insulin and 15 and 45 days for rH insulin (Fig. 2A) compared to 135 and 160 days for bovine (Upper) and rH (Lower) insulin, respectively, when 200 g SIA-II was used. Dosage of 400 g resulted in sudden hypoglycemia, suggesting an initial bolus release (above basal level) of insulin monomers at this dose (Fig. 2A). Thus 200 g SIA-II (both bovine and rH insulin) was chosen as a therapeutic dosage for the detailed long-term prospective studies. Preprandial (overnight 8- to 10-h fasting) and postprandial (nonfasting/fed state, i.e., random-fed BGL taken at 11.00 AM). Blood glucose levels (BGL) were monitored till the physiological effect of SIA-II was observed. Treatment of STZ-induced diabetic rats with 200 g of SIA-II led to a significant reduction in the nonfasting BGL (Fig. 2B) without fasting hypoglycemia (Fig. 2C). No significant difference was observed whether SIA-II was injected subcutaneously or intramuscularly. Daily insulin dosage (4 unitskg) led to reduced BGL, nonfasting value of 350450 mgdL, and fasting value of 300400 mgdL for bovine insulin and 350 400 mgdL (random-fed state) and 240260 mgdL (fasting) for rH insulin. Twice daily injection of 4 unitskg insulin (bovine and rH insulin) marginally improved the glycemic levels. While
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Fig. 2. In vivo efficacy of SIA-II (both bovine and rH) in glucose homeostasis. (A) Preprandial/fasting blood glucose profile of diabetic animals in response to different SIA-II dosages. (B) Postprandial BGL and (C) preprandial BGLs of nondiabetic rats (control) and diabetic rats treated with PBS-200 L; insulin single dose-4 unitskg body weight (b.wt) in the morning; insulin double dose- 4 IUkg b.wt in the morning and evening; SIA-II (SC, IM)- 200 g of SIA-II administered either s.c. or i.m., monitored over a period of 135 days for bovine (Left) and 160 days for rH (Right) SIA-II in diabetic rats. n 10 animals in each group. P < 0.001 STZ vs. insulin/SIA-II; insulin vs. SIA-II, P 0.462 fasting control vs. SIA-II.

the daily dose of insulin was able to maintain glucose levels at moderately high values, a single injection of SIA-II insulin was sufficient for achieving near-normoglycemic postprandial levels (201 25.96 mgdL and 206 38.68 mgdL in s.c. and i.m., respectively) in diabetic rat for about 135 days without fasting hypoglycemia (124 11.82 mgdL and 130 12.9 mgdL in s. c. and i.m., respectively) where the oligomer was made of bovine insulin. Administration of rH insulin SIA-II resulted in a more desirable glycemic control with BGL of 160 13.61 mgdL (fed state) and 105 15 mgdL (fasting) for up to 160 days with a single injection (both s.c. and i.m.) (Fig. 2 B and C). BGL for the initial 6 days for both bovine and rH SIA-II are given in SI Text (Fig. S2 CF). Body weight, which is an indicator of normal health, was monitored over the entire period of treatment. There was an initial loss of body weight immediately after STZ injection (Fig. 3A and Fig. S2B), but progressive weight gains were achieved in diabetic rats after treatment with insulin SIA-II, and the curve of SIA-II treated animals paralleled that of nondiabetic control. Thus, the striking efficacy of insulin SIA-II therapy as a long-term treatment of DM-1 in diabetic animals over that observed for the conventional therapy constitutes a key finding of this study.
Glucose Tolerance Test (GTT). GTT was performed to assess the instantaneous release of insulin monomers from SIA-II in vivo, from the site of injection. The biological activity of the released monomer was estimated by blood glucose disposal from the blood. Bovine insulin, SIA-II, and in vitro released monomers, significantly improved the glucose tolerance in treated animals, with their elevated BGL of 600 mgdL due to glucose infusion returning to the prechallenged values within 1.5 h. However, in diabetic controls treated with PBS, BGL remained hyperglycemic over the experimental period (Fig. 3B). Serum Insulin Level. Optimum BGL were sustained for a period of about 135 days for bovine insulin and 160 days for rH insulin, with a single dose of the insulin SIA-II. From the day of injection and throughout the experimental period, a sustained level of rH/
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bovine insulin (0.51.2 ngmL) into the serum was observed (Fig. 3C) compared to endogenous level of rat insulin (0.08 ngmL) in PBS-treated diabetic rats. Serum bovine insulin levels were also quantified for GTT (Fig. 3D), which were expectedly negligible for both diabetic and nondiabetic controls, as no SIA-II was administered to these. In contrast, the values for native insulin and in vitro released monomers from SIA-II increased to 0.9 ngmL in 30 min and then decreased in the next 4.5 h. In SIA-II treatment (s.c.), insulin levels in serum

Fig. 3. (A) Body weight profile of SIA-II treated rats. (B) Blood glucose profile of GTT. (C) Quantification of serum (bovine or recombinant human) insulin ELISA in STZ-treated rats in response to insulin (bovine or rH) SIA-II injected s.c. or i.m.. (D) Quantification of serum bovine insulin of GTT. Results are mean SD of three different experiments. n 10 animals in each group. n 5 animals in each group. P < 0.001 STZ vs. insulin/SIA-II/released, P 1.0 insulin vs. released insulin from SIA-II, P 0.234 control vs. SIA-II. P 0.548 insulin level (s.c.) vs. (i.m.), P < 0.05 STZ vs. insulin/ SIA-II, P 0.002 control vs. STZ.
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Fig. 4. 125 I labeled bovine insulin SIA-II treatment of STZ-diabetic rats. (A) cpmmL profile in serum of animals treated with 100 g of labeled SIA-II up to 25 days. The profile for 24 h is given in the inset. (B) Fed blood glucose (mgdL) and bovine insulin (ngmL) released in vivo from SIA-II profile for 36 days. The profile for 24 h is shown in the inset. (C) Tricine-SDS-PAGE of serum from animals treated with 125 I labeled SIA-II. Coommassie staining (Left) and phosphor images (rest after left) of serum of 128 days after treatment either subcutaneously (Upper) or intramuscularly (Middle). (Lower) Insulin monomers released from 125 I-SIA-II in vitro. (D) Gel chromatogram of insulin, released insulin from SIA-II and tagged insulin. (E) SDS-PAGE gel (lane 1, cross-linked released insulin; lane 2, cross-linked insulin; lane 3, marker; lane 4, released insulin; and lane 5, rH insulin). Results are mean SD of three different experiments having n 5 animals in each group. I, insulin monomer; M, molecular weight marker.

125 I Labeling of Insulin. To further validate and quantitate the in vivo release of insulin, SIA-II formed from 125 I-labeled bovine insulin with a specific activity of 499;122 CPMg was injected either s.c. or i.m. CPMmL in serum of treated animals remained nearly constant (Fig. 4A). An initial high count was observed between 0.54 h, which gradually decreased to a constant level of 2;0003;000 cpmmL in 10 h. The blood glucose profile was the same as observed with the unlabeled SIA-II (Fig. 4B). Serum insulin levels quantified using ELISA were in the range of 0.51.2 ngmL, which correspond to basal or a moderately above basal level of insulin (Fig. 4B). To further investigate the amount of the released insulin from SIA-II, serum samples of different time points were resolved on Tricine-SDS-PAGE (Fig. 4C). Bands obtained on the phosphor image correspond to free insulin and its intensity remained constant when an equal amount of serum was loaded. The monomeric nature of insulin released from SIA-II was also validated using size exclusion chromatography (Fig. 4D) and glutaraldehyde cross-linking (Fig. 4E), where the released insulin band corresponded to that of native insulin monomer.

reached 0.80.9 ngmL in 30 min and were sustained over the period of study (Fig. 3D). Thus insulin is released instantaneously from the depot formed at the site of injection.

Insulin Signaling. The effect of insulin on glucose transport and other metabolic events in adipose tissues are mediated by intracellular signaling cascades (PI3K, AKT, GSK3, and ERK) (4), which start after insulin monomers bind to insulin receptors on the cell surface. The biological activity of monomers released from SIA-II was evaluated using isolated rat adipocytes. Levels of PI3K, which is the first kinase downstream to insulin signaling via its receptor, increased significantly in response to SIA-II (bovine and rH insulin), insulin (bovine and rH insulin), and insulin released in vitro (from SIA-II) as compared to PBS control (Fig. 5A). A significant increase in the phosphorylation of Akt, a threonine/serine kinase important for insulin regulation and various metabolic responses in adipocytes, was observed,

Fig. 5. Western blot (WB) analysis of insulin and IGF-1R signaling cascade: (A) adipocytes treated with PBS (control), insulin-human (Insulin-H), insulin-bovine (Insulin-B), SIA-II from rH insulin (SIA-H), SIA-II from bovine insulin (SIA-B), and released monomers from SIA-II (Released Ins). (B) Adipocytes treated with serum of insulin and SIA-II treated rats and analyzed for insulin signaling (WB). (C) Competitive ligand binding to IGF-1R (overexpressed in NIH-3T3), with 100 pM 125 I-IGF-1 and various concentration of unlabeled IGF-1, insulin, and released insulin from SIA-II, as described in the Materials and Methods. (D) Western blot analysis of IRS-1/2 phosphorylation upon stimulation of IGF-1R with PBS (control), only insulin (rH-insulin, 20 nM), SIA-II (20 nM), released insulin from SIA-II (R-insulin, 20 nM), and IGF-1 (6 ngmL). Histology of subcutaneous sections from diabetic rats treated with insulin SIA-II (pH 7.0) or fully formed amyloid fibrils at pH 2.0: binding of CR to subcutaneously (40X) injected SIA-II was examined [E(iv)]. CR birefringence (BF) was observed under plane polarized light in the above mentioned same s.c. [E(vix)] sections. Inflammation caused by the infiltration of cells was examined by H&E staining, and the presence of neutrophils, myeloid lineage cells, and NK cells was detected by immunostaining against CD11b, RT-1A, and CD6 antigens in s.c. and i.m. tissue sections. Absence of inflammation seen in s.c. [E(xixv) and E(xvixx)] tissue sections, observed by immunostaining and H&E staining, respectively, as compared to positive control of inflammation shown by injection of LPS to rats (neutrophil infiltration stained with CD11b), both subcutaneously [F(i & iii)] and intramuscularly [F(ii and iv)] (100X). CR bound to pH 2.0 amyloid in s.c. sections [G(iiv)] monitored for a period of 12 weeks and corroborated with CR birefringence [G(vviii)]. Results are mean SD of three different experiments done in triplicates.
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similar to the response obtained for free insulin. Phosphorylation of GSK3 on Ser-21 increased several fold in adipocytes treated with either free insulin or SIA-II compared to control (Fig. 5A). Therefore, both Akt and GSK3 showed rapid and pronounced response to SIA-II, and the results were similar to responses observed on stimulation with free insulin (used as positive control). A significant activation of ERK1/2 was observed compared to PBS control in case of treated adipocytes. Similar activation of signaling mediators was observed, when serum from insulin or its SIA-II-treated animals was added to cultured adipocytes (Fig. 5B).
Insulin-Like Growth Factor 1 Receptor (IGF-1R) Binding Studies. Due to the structural similarity of insulin with insulin-like growth factor 1 (IGF-1), it is imperative to study the affinity of insulin released from the SIA-II toward binding to IGF-1R. Altered (i.e., increased) affinity may result in the activation of mitogenic response upon binding. To exclude such a possibility and confirm that the released monomers from the SIA-II are unaltered insulin monomers, the affinity of rH insulin, SIA-II, and IGF-1 to bind to IGF-1R was studied. NIH-3T3 cells overexpressing the human IGF-1R were utilized to perform the competition binding assay with both labeled (125 I) and unlabeled rH insulin and IGF-1 and analyzed by Scatchard plot. As evident from Fig. 5C, the ED50 of IGF-1 binding to IGF-1R was determined to be 1.00 0.216 nM, whereas that of insulin and insulin released from SIA-II was 0.134 0.021 M and 0.175 0.03 M, respectively. Low concentration of IGF-1 was able to displace 125 IGF-1, in contrast to high concentration of insulin and released insulin. Thus the affinity of released insulin from the SIA-II for IGF-1R is not altered. This was also validated by Western blot, where the differential phosphorylation profile of IRS-1/2 was similar for both insulin and insulin released from SIA-II (less phosphorylation of IRS-1), and is distinct from that observed for IGF-1 (more IRS-1 phosphorylation) (Fig. 5D). IRS-1 is preferentially phosphorylated upon binding of IGF-1 to IGF-1R, whereas IRS-2 phosphorylation is more when insulin binds to insulin receptor. Ligand binding to IGF-1R was completely obliterated in the presence of IGF-1R antibody, but retained when antibody to insulin receptor was used (Table S1). CR, H&E, and Immunostaining of Subcutaneous and Muscle Tissues.

16 weeks of treatment with SIA-II [Fig. S3B(ivi)], as these tissues maintained normal cellular morphology [Fig. S3B(viiix)].
Reduction of Secondary Complications Related to Hyperglycemia in Type 1 Diabetes. Type 1 diabetic rats show a marked decrease

in skeletal muscle (2040%) and abdominal fat (>60%) (3, 5). In contrast, SIA-II-treated diabetic animals had normal body weight and were healthy. Developments of cataract in insulintreated diabetic rats as well as in untreated diabetic rats were seen, whereas no cataract was observed in animals treated with SIA-II (Fig. S3C). Moreover, functions of liver and kidney, the two main organs of the body that typically and severely get affected in diabetes, were almost normal in SIA-II-treated rats as compared to their untreated or free insulin infused counterparts (Table S2).
Absence of Insulin Degrading Enzyme (IDE) and Screening of Antibodies Against Insulin. Subcutaneous resistance to insulin in DM-1

diabetes is a rare syndrome (6), defined as the lack of biological activity of subcutaneously injected insulin; nevertheless, efficacy of intravenously infused insulin is retained. It has been reported that IDE degrades insulin specifically (7) and the partially degraded insulin is reabsorbed into the circulation with increased immunogenicity but no biological activity. To check the development of such a resistance in insulin SIA-II-treated animals, biological activity of IDE and the presence of antiinsulin antibodies in serum were tested. IDE activity was found to be negligible and comparable to that of normal rats in tissue homogenate of SIA-II treated animals (Fig. S4A). Similarly antiinsulin antibodies were not observed even after 16 weeks of treatment, supporting the nonimmunogenecity of insulin SIA-II and the absence of IDE induction (Fig. S4B). Discussion In DM-1, maintenance of normal glycemic levels throughout the day is a great concern, which is not fulfilled efficiently by the existing therapies. Patients with DM-1 need insulin once or twice a day to sustain normoglycemia. In addition to this, postprandial glucose homeostasis is maintained through regular insulin injections before each meal. Intensive insulin therapy delays the onset and/or slows the progression of secondary complications. Longacting insulin analogues, such as glargine and detemir, achieve more constant glucose levels but suffered a setback due to induction of fasting hypoglycemia (8). Therefore, a formulation that releases insulin in a controlled manner for longer periods of time without any burst or rapid release would free the patients from the need to administer multiple doses of insulin. We employed the remarkable properties exhibited by the insulin oligomer, such as increased stability, protease resistance, and longer shelf life, to develop an insulin prodrugsupramolecular insulin assembly II, which releases the hormone in a controlled fashion for extended periods of time and can be harnessed for therapeutic purposes. SIA-II is a prodrug, where neither any chemical modification was done for its formation in vitro nor is chemical cleavage required for achieving its therapeutic effect in vivo. The intrinsic property of the protein to unfold and oligomerize has been harnessed here to achieve a glucose regulatory effect. Moreover, the period of sustained release is 100 times greater than any of the studies reported earlier (9). SIA-II selected by us was observed to be a linear association of elongated oligomers by AFM, which released insulin monomers at a constant rate. The SIA-II and SIA-III were resistant to protease cleavage by proteinase K, in comparison to insulin and SIA-I, which were more susceptible. The chosen intermediate did not show significant CR binding and corresponded to a sharp IR band position at 1;6471;645 cm1 , unlike the amyloid state of protein.
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Diabetic rats treated with SIA-II and amyloid formed at pH 7.0 and 2.0 were monitored for the presence of the depot and the residual amount of SIA-II or amyloid for 1, 2, 4, 8, and 16 weeks using CR staining. Tissues obtained after 1 week of SIA-II (pH 7.0) injection were found to bind CR, confirming the presence of higher amounts of SIA-II [Fig. 5E(i) and Fig. S3A(i)]. The amount of bound CR thereafter decreased in a time-dependent manner [Fig. 5E(iiiv) and Fig. S3A(iiiv)] and was considerably lower after 16 weeks [Fig. 5E(v) and Fig. S3A(v)], corroborated with a birefringence of bound CR [Fig. 5E(vix) and Fig. S3A(vix)]. The same tissues were also checked for inflammation caused, if any, due to insulin SIA-II by H&E and immunostaining. As compared to the sections from LPS-injected rats, where infiltration of proinflammatory cells were visible in both subcutaneous [Fig. 5F(i) and (iii)] and muscle tissues [Fig. 5F(ii) and (iv)], SIA-II-injected sections did not show significant inflammation even after 16 weeks in both immunostained [Fig. 5E(xvixx) and Fig. S3A(xixv)] and H&E [Fig. 5E (xixv)] sections, confirming the noninflammatory and nonimmunogenic nature of the SIA-II being used. As described earlier, amyloid fibrils formed at pH 2.0 bound to CR very efficiently and its intensity reduced negligibly over the course of our study, validating the absence of insulin release from these mature fibers [Fig. 5G(iviii)]. The possibility of deposition of higher oligomers of injected SIA-II in heart, kidney, and liver was ruled out by the insignificant CR binding to these tissue sections even after
Gupta et al.

APPLIED BIOLOGICAL SCIENCES

The biological effectiveness of insulin is generally assessed by its ability to regulate glycemic levels in the blood. Resultant decrease in the blood glucose concentration represents the most noticeable and therapeutically the most important effect of insulin. As hypothesized, SIA-II treatment reduced the severity of hyperglycemia and a single dose of 200 g maintained near-normoglycemic levels (160 13.6 mgdL) in diabetic animals for about 120140 days. To determine whether improvement in glycemic control by SIA-II was accompanied with increased risk of fasting hypoglycemia, treated animals were subjected to weekly extended fasting of 1820 h (Fig. S5 A and B). These fasted animals were able to maintain their fasting BGL within the normal range (60110 mgdL). Thus in comparison to conventional insulin therapy, SIA-II treatment achieved a tightly regulated glycemic control without fasting hypoglycemia. GTT conducted to determine the onset of the action of insulin SIA-II in case of induced severe hyperglycemia demonstrated no lag phase in the release of bioactive insulin monomer from the SIA-II depot. Continuous subcutaneous insulin infusion using insulin pump therapy had been developed earlier to overcome the problem of multiple daily injections and was fairly successful in maintaining BGL (10). However, the extra cost of the pump, pump malfunctions, bacterial infections in subcutaneous catheters, delayed and unpredictable absorption from the subcutaneous site, variability of insulin sensitivity, diabetic gastroparesis, and need of trained personnel to supervise added to the woes of this therapy (11, 12). To assess the in vivo release of human recombinant insulin from SIA-II, ELISA was performed. A sustained release of insulin (0.50.9 ngmL) sufficient for maintaining normoglycemia was seen, which correlated well with the duration of the treatment. Radiolabeling of insulin with 125 I was done to validate the release kinetics of monomers from insulin. Amount of insulin quantified by ELISA in the serum of treated rats was comparable to the labeled insulin released in the serum upon administration of 499;122 CPMg of SIA-II. Constant release of insulin, with no burst phase, from the depot formed at the site of injection follows zero-order kinetics, a beneficial requirement for the sustained and extended period of treatment seen with SIA-II. Radioactive serum samples resolved onto Tricine-SDS-PAGE showed bands corresponding to insulin monomer, which was also confirmed by size exclusion chromatography and glutaraldehyde cross-linking studies. These monomers triggered insulin signaling cascade efficaciously by the activation of intracellular mediators (PI3K, Akt, GSK3, and ERK1/2) in cultured adipocytes, suggesting that the monomers released from SIA-II are biologically active. Storage did not alter the blood glucose lowering effect of SIAII. Slow release of insulin was again ascertained by histological studies of tissues from rats injected with SIA-II, which also demonstrated the formation of a depot at the site of injection from which insulin is released over a period of time and resulted in a decrease of the residual SIA-II. No inflammation was observed at the site of injection even after 16 weeks. The SIA-II therapy conferred profound physiological benefits in diabetic animals, which partially reflected an improved
1. Brown A, et al. (2010) Intensive glycemic control and cardiovascular disease: An update. Nat Rev Cardiol doi: 10.1038/nrcardio.2010.35. 2. Burn Paul (2010) Type 1 diabetes. Nat Rev Drug Discov 9:187188. 3. Smith JF, et al. (2006) Characterization of the nanoscale properties of individual amyloid fibrils. Proc Natl Acad Sci USA 103:1580615811. 4. Paul Bevan (2001) Insulin signalling. J Cell Sci 114:14291430. 5. Nathan DM, et al. (2005) Intensive diabetes treatment and cardiovascular disease in patients with type 1 diabetes. N Engl J Med 353:264353. 6. Paulsen EP, et al. (1979) Insulin resistance caused by massive degradation of subcutaneous insulin. Diabetes 28:640645. 7. Duckworth WC, et al. (1998) Insulin degradation: Progress and potential. Endocr Rev 19:608624.

glycemic control as well as markedly reduced urea and creatinine concentrations due to improved liver and kidney functions, respectively. Absence of antiinsulin antibodies and inability to elicit IDE in or around the site of injection are important factors in prolonged beneficial antidiabetic affect. The report presented here shows that insulin released from SIA-II is equivalent to soluble insulin as far as its biological function is concerned. A significant difference lies in the duration of action. Wheras for the standard insulin injection, it is only 610 h (7), for SIA-II the duration of action is markedly enhanced up to 135 days for bovine and 160 days for rH insulin. This may be attributed to the remarkable stability of insulin in the SIA-II state, which forms a depot at the site of injection for supply of the physiologically most relevant form of insulin, namely, insulin monomers, for a longer period of time. In contrast, any peptide or protein drug administered is cleared and/or degraded from the body at a faster rate, thereby decreasing the window period of its efficacy. Studies with SIA-II as a therapeutic for diabetes type 1 was also carried out in other model systems such as in mice and rabbit and also using the Alloxan for induction of diabetes apart from STZ (Fig. S5 C and D). Similar beneficial outcomes were noted in these models of diabetes also (Fig. S6 AD). In summary, we have demonstrated the efficacy as well as the feasibility of insulin SIA-II treatment for the significant improvement of BGL without causing fasting hypoglycemia in STZ-induced diabetic animal model. Unlike intensive insulin therapy, by which BGLs are controlled by an increased frequency of insulin injection with concomitant risk of hypoglycemia (3), the significantly improved glycemic control using insulin SIA-II therapy is accomplished without multiple insulin injections, which displays no adverse side effects. Materials and Methods
Insulin Fibrilization. Bovine or recombinant human insulin (Sigma), 2 mgmL (28 unitsmg), was dissolved in PBS at pH 7.0 or hydrochloric acid in water (pH 2.0) and incubated at 37 C for 7 days with constant agitation at 180 rpm. The process of oligomerization was monitored by turbidity at 600 nm and by Th-T fluorescence. In Vitro Monomer Release Kinetics. Aliquots of 200 L were withdrawn at different time points from the insulin fibrilization reaction at pH 7.0 and 2.0, 37 C. Insulin oligomers produced at different time points were isolated by centrifugation at 10,000 rpm for 10 min. The pellet obtained was washed with PBS and resuspended in 1 mL PBS in an Eppendorf tube, cap was removed, and the Eppendorf was sealed with 8-kDa cutoff dialysis membrane. The Eppendorf tube was suspended in 20 mL PBS containing 0.02% sodium azide in a 50-mL Falcon tube and the kinetics of insulin release into PBS was monitored spectrophotometrically at 280 nm and by its intrinsic (tyrosine) fluorescence for 15 days. The amount of insulin released per hour was calculated. A complete and detailed description of the methods is included in SI Text. ACKNOWLEDGMENTS. The authors thank Dr. Ira Surolia and Dr. Shiv Pillai (Massachusetts General Hospital) for a critical reading of the manuscript.
8. Hirsch B (2005) Insulin Analogues. New Eng J Med 352:174183. 9. Gershonov E, et al. (2000) A novel approach for a water-soluble long-acting insulin prodrug: Design, preparation, and analysis of [(2-sulfo)-9-fluorenylmethoxycarbonyl](3)-insulin. J Med Chem 43:25307. 10. Pickup JC, Renard E (2008) Long-acting insulin analogs versus insulin pump therapy for the treatment of type 1 and type 2 diabetes. Diabetes Care 31:S140S145. 11. Nowakowska M, et al. (2007) Bacterial strains colonizing subcutaneous catheters of personal insulin pumps. Pol J Microbiol 56:23943. 12. Shalitin S, Phillip M (2008) Hypoglycemia in type 1 diabetes: A still unresolved problem in the era of insulin analogs and pump therapy. Diabetes Care 31(Suppl 2):S121S122.

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Gupta et al.